Evolution of class-I MHC genes and proteins: from natural selection to thymic selection.
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The level of CD8 expression can determine the outcome of thymic selection.
During thymic development, thymocytes that can recognize major histocompatability complex (MHC) molecules on thymic epithelial cells are selected to survive and mature (positive selection), whereas thymocytes that recognize MHC on hematopoietic cells are destroyed (negative selection). It is not known how MHC recognition can mediate both death and survival. One model to explain this paradox proposes that thymocytes whose T cell antigen receptors (TCRs) recognize MHC with high affinity are eliminated by negative selection, whereas low affinity TCR-MHC interactions are sufficient to mediate positive selection. Here we report that, while the expression of a 2C TCR transgene leads to positive selection of thymocytes in H-2b mice, expression of both a CD8 transgene and a 2C TCR transgene causes negative selection. This observation indicates that quantitative differences in TCR-MHC recognition are a critical determinant of T cell fate, a finding predicted by the affinity model for thymic selection.
Animals↗
Influence of the major histocompatibility complex on positive thymic selection of V beta 17a+ T cells.
A monoclonal antibody was used to show directly positive thymic selection of the T cell repertoire in mouse strains expressing the 17a beta-chain variable domain (V beta 17a) of the T cell receptor. In the absence of the potent tolerizing class II major histocompatibility complex (MHC) molecule, I-E, peripheral expression of V beta 17a+ T cell receptors varied with the MHC haplotype of the mouse strain. In the most extreme case, H-2q mice expressed high peripheral levels of CD4+ V beta 17a+ T cells (14 to 19 percent), whereas H-2b mice expressed low levels (3 to 4 percent). Analysis of (b x q)F1 mice and chimeric mice showed that these differences were determined by positive thymic selection and implicated the thymic epithelium as the controlling cell type.
Animals↗
A unified model for T cell antigen recognition and thymic selection of the T cell repertoire.
Positive selection of T cells during thymic differentiation predisposes mature T cells to recognize glycoproteins encoded by "self" alleles of the major histocompatibility complex (MHC) as "restricting elements" for antigen presentation. Yet, negative selection also occurs during thymic differentiation resulting in the clonal deletion of T cells reactive with "self" MHC glycoproteins. Thymic processes of positive and negative selection represent a paradox because the "altered self" view of T cell receptor (TcR) recognition indicates that the same type of TcR-MHC glycoprotein binding interaction mediates both positive and negative selection of the T cell repertoire. Most contemporary models of thymic selection attempt to explain these paradoxical observations by quantitative differences of affinity. That is, TcR interactions with MHC ligands that are of medium affinity lead to positive selection whereas those that are of high affinity lead to negative selection. The purpose of this manuscript is to provide an alternative model of thymic selection based on the efficacy (the ability of a ligand to catalyze receptor mediated biological activity) of TcR-MHC ligand interactions. The "efficacy" model predicts that among those thymocytes exhibiting affinity for self MHC ligands, some clones bind self MHC ligands without efficacy whereas others bind these ligands with efficacy. Immature T cells that bind MHC ligands without efficacy do not undergo TcR mediated activation and thereby escape clonal deletion. Instead, these T cells compete for growth--promoting sites on thymic antigen presenting cells (APC) based upon their clonotypic TcR affinity for self MHC ligands. These T cells experience positive thymic selection and eventually dominate a repertoire of mature T cells predisposed to exhibit non-efficacious binding to "self" MHC ligands. In contrast, immature T cells that exhibit efficacious binding to self MHC ligands are deleted from the T cell repertoire during thymic maturation. By this mechanism, the mature T cell repertoire is selected so that clonotypic T cells are predisposed to bind the very sites on MHC glycoproteins responsible for antigen presentation without risk of autoimmunity. Given the clonotypic diversity of the mature repertoire, complexes of foreign peptides and self MHC glycoproteins would be recognized by clones of the appropriate specificity as highly efficacious ligands. In summary, the "efficacy" model is entirely consistent with the "altered self" concept of T cell antigen recognition and readily accounts for both positive and negative selection of the T cell repertoire.
Antigens, CD↗
Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.
Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte-binding protein, T11, represents an essential cell surface component of a human T-cell lineage activation pathway. Furthermore, it is known that the human T3-Ti T-cell antigen/major histocompatibility complex receptor complex is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2-receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and must possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.
Antigens, Differentiation, T-Lymphocyte↗
Selective inhibition of interleukin 2 gene function following thymocyte antigen/major histocompatibility complex receptor crosslinking: possible thymic selection mechanism.
Considerable evidence now exists to support the notion that the 50-kDa sheep erythrocyte binding protein, T11, represents an essential cell surface component of a human T-cell-lineage activation pathway. Furthermore, it is known that the human T-cell antigen-major histocompatibility complex (MHC) receptor complex T3-Ti is capable of regulating cell growth mediated by the T11 structure. Here we show that, within the T3+ thymocyte compartment, T3-Ti crosslinking rapidly inhibits T11-initiated interleukin 2 (IL-2) gene transcription and translation. This inhibition is restricted to the IL-2 gene (IL2) as transcription of both the IL-2 receptor gene (IL2R) and the Ti beta-chain gene (TCRB) are not affected (human gene designations are in parentheses). Perhaps more importantly, T3-Ti-mediated IL-2 inhibition of this type is not operational in peripheral T lymphocytes. The results imply that the majority of T3+ thymocytes are functionally distinct from peripheral T lymphocytes despite their T3+ phenotype and may possess a unique endogenous regulatory component for suppressing IL-2 gene activity. Moreover, since IL-2 is likely rate-limiting for growth within the thymus, the findings provide one plausible mechanism for thymic selection--namely, T3-Ti crosslinking of thymocytes upon interaction with self-major histocompatibility complex inhibits clonal expansion of high-affinity autoreactive cells.
Antigens, Differentiation, T-Lymphocyte↗
Thymic selection of the human T cell receptor V beta repertoire in SCID-hu mice.
Implantation of pieces of human fetal liver and thymus into SCID mice results in the development of a human thymus-like organ, in which sustained lymphopoiesis is reproducibly observed. In this model, T cell development can be experimentally manipulated. To study the influence of thymic selection on the development of the human T cell repertoire, the T cell receptor (TCR) V beta gene repertoire of double-positive (CD4+CD8+) and single-positive (CD4+CD8- and CD4-CD8+) T cells was analyzed in the SCID-hu thymus using a multiprobe ribonuclease protection assay. TCR diversity in double-positive SCID-hu thymocytes was found to be comparable with that present in the thymus of the fetal liver donor, did not change with time, and was independent of the origin of the thymus donor. Thymic selection in SCID-hu thymus induces changes in V beta usage by the single-positive CD4+ or CD8+ T cells comparable with those previously reported for single-positive cells present in a normal human thymus. Finally, significant differences were observed in the V beta usage by CD4 or CD8 single-positive T cells that matured from genetically identical stem cells in different thymic environments. Collectively, these data suggest: first, that the generation of TCR diversity at the double-positive stage is determined by the genotype of the stem cells; and second, that polymorphic determinants expressed by thymic epithelium measurably influence the V beta repertoire of mature single-positive T cells.
Alleles↗
CD45RA and CD45RBhigh expression induced by thymic selection events.
CD45 is a protein tyrosine phosphatase involved in T and B cell signaling. While peripheral T cells switch CD45 isoforms upon activation, events leading to exon switching during T cell development in the thymus have not been determined. The expression of high molecular weight isoforms of CD45 was examined on thymocytes from nontransgenic and T cell receptor (TCR) transgenic mice. All thymocytes from nontransgenic mice were CD45RB+ as assessed by staining with MB23G2, an anti-CD45RB-specific monoclonal antibody. Interestingly, there was a small population (1-3%) of thymocytes that displayed a higher intensity of staining with MB23G2, CD45RBhigh. CD45RBhigh thymocytes were found in all subsets defined by CD4 and CD8 expression and were also present within the TCR-alpha/beta high population. To analyze whether or not CD45 expression correlated with thymic selection events, expression of CD45RBhigh and a second isoform, CD45RA, was examined on thymocytes from H-Y and 2C TCR transgenic mice and found to correlate with positive and negative selection events but did not occur in nonselecting backgrounds. CD45RA and CD45RBhigh upregulation was also not observed in transgenic mice backcrossed into CD8-deficient mice, a scenario in which there is no positive selection of transgene-expressing thymocytes. These data suggest that modulation of CD45 isoform expression may be involved in thymic selection events.
Animals↗
Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage.
Immature thymocytes, which coexpress CD4 and CD8, give rise to mature CD4+CD8- and CD4-CD8+ T cells. Only those T cells that recognize self-MHC are selected to mature, a process known as positive selection. The specificity of the T cell antigen receptor (TCR) for class I or class II MHC influences the commitment to a CD4 or CD8 lineage. This may occur by a directed mechanism or by stochastic commitment followed by a selection step that allows only CD8+, class I-specific and CD4+, class II-specific cells to survive. We have generated a mouse line expressing a CD8 transgene under the control of the T cell-specific CD2 regulatory sequences. Although constitutive CD8 expression does not affect thymic selection of CD4+ cells, selection of a class I-specific TCR in the CD8 subset is substantially improved. This outcome is consistent with a model for positive selection in which selection occurs at a developmental stage in which both CD4 and CD8 are expressed, and positive selection by class I MHC generates an instructive signal that directs differentiation to a CD8 lineage.
Animals↗
Imprint of thymic selection on autoreactive repertoires.
We have focussed on the differences in origin and physiological properties of two classes of self-reactive T cells. Autoreactive T cells described in many laboratories are activated in the course of normal immune responses to foreign antigen. These T cells can be shown under well-defined conditions to be the direct progeny of antigen-stimulated precursors. This, together with evidence that their activation requirements can be distinguished from those of antigen-specific, MHC-restricted T cells, leads us to suggest that they represent a particular physiological state that recapitulates the conditions of thymic selection and is induced in many antigen-specific, MHC-restricted peripheral T cells as a result of normal antigen-dependent activation. Although it appears that the associated physiological properties can be stable in some in vitro maintained lines, it is possible that this is normally a transient state in vivo. Available evidence concerning the specificity of these T cells indicates only that they can be activated in the absence of any identifiable foreign antigen by class II MHC-syngeneic but not MHC-allogeneic stimulators. We have suggested that such T cells are specific for the same elements, possibly an association of MHC and other self-peptides (Singer et al. 1987), that are the basis for positive selection in the thymus. The properties of these autoreactive T cells need to be distinguished from those of T cells associated with autoimmune pathology. It is presumed that autoimmune T cells are directly activated in a resting state by specific self-peptides. Our interest in distinguishing these self-reactive T-cell populations has focussed on different predictions concerning the diversity of their associated self-reactive repertoires. The relative complexity of the immune repertoire expressed in autoreactive T cells expanded by positive selection and restimulated in the course of normal antigen-specific immune responses should be considerably greater than that of autoimmune T cells constrained by negative selection and a narrow window of escape from self-tolerance. We were greatly hindered in our initial efforts in this analysis by the considerable effort required to characterize any specific immune repertoire. A published technique employing poly(A) tailing (Frohman et al. 1988) did not work efficiently in our hands, although others (Loh et al. 1989) have apparently had some success. We describe above an alternative approach, linker-facilitated PCR, which we have employed for efficient repertoire analysis. Using this method we have been able to identify dominant utilization of the Va4 family in T cells specific for the synthetic peptide YYEELLKYYEELLK.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Selective thymic localization of murine leukemia virus-related antigens in C57BL-Ka mice after inoculation with radiation virus.
The tissue distribution and extent of virus-specific antigen expression were studied by immunofluorescence as a function of time and of lymphoma development in adult C57BL/Ka (Fv-1(b)) mice after intravenous injection of radiation leukemia virus, a B-tropic murine leukemia virus. Viral antigens were detected earlier in the thymus (1 week) than in the bone marrow, spleen, or lymph nodes (2-3 weeks). Despite an initial virus-induced thymic involution, the percentage of immunofluorescence-positive cells in the thymus rapidly increased thereafter to 65-80%, at which level it remained until 9 weeks, at which time increases in size and weight, histological changes, and an increased number of blastoid cells indicated the onset of lymphoma development in the thymus. In contrast, the percentage of immunofluorescence-positive cells in the bone marrow, spleen, and nodes remained low, and gradually decreased to zero within 8 weeks after thymectomy. The selective thymic localization of antigens induced by radiation leukemia virus in C57BL/Ka mice is in striking contrast to the previously reported ubiquitous tissue distribution of the Gross-AKR virus, an N-tropic virus, in its natural host, the Fv-1(n), AKR strain with a high incidence of leukemia.
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Expression of identical V alpha V beta gene pairs by IE-alloreactive and IE-restricted, antigen-specific T cells from MHC disparate mice. Evidence for thymic selection of V(D)J combinations.
Alloreactive T cell hybridomas specific for IEk and/or IEb MHC Ag were obtained from IE-nonexpressor (IE alpha b) mice. The TCR V alpha and V beta gene segments used were identified by Northern blot and RNase protection. A large proportion (24 of 80 hybridomas tested) employed the same V alpha genes (V alpha 11.1 or V alpha 11.2) as are utilized in the IEk and IEb restricted response to the Ag cytochrome c. Of these 24 alloreactive hybridomas, 10 also expressed V beta genes utilized in the IE plus cytochrome c repertoire. Structural similarity between the two related sets of TCR indicates that V alpha segments can play a determining role in MHC specificity. These data also suggest that thymic selection based on TCR reactivity with self-MHC products acts on particular V(D)J combinations rather than on V alpha V beta pairings alone.
Animals↗
Thymic selection of H-2-incompatible bone marrow cells in SCID mice. Differences in T help for induction of B cell IgG responses versus cytotoxic T cells.
Mice with congenital severe combined immunodeficiency disease (SCID) failed to mount either a T cell-independent IgM or T cell-dependent IgG anti-vesicular stomatitis virus (VSV) Indiana (IND) response. They did not generate cytotoxic T cells against lymphocytic choriomeningitis virus (LCMV) or vaccinia virus, but exhibited NK cell-like activities. When SCID mice were given bone marrow from syngeneic BALB/c (H-2d) nu/nu mice, all immune responses were expressed at control levels. If SCID mice were reconstituted with allogeneic H-2b C57BL/6 nu/nu bone marrow, the following primary anti-viral immune responses were measured. T-independent IgM anti-VSV-IND were normal, but T-dependent IgG anti-VSV-IND responses were absent. Cytotoxic T cell responses to LCMV and vaccinia virus were within normal ranges, were donor cell mediated, and were specific exclusively for the recipient SCID H-2d type. Since antigen presentation by spleen cells was functional in these chimaeras, the presented results indicate that (a) thymic selection of T cell restriction is strict; and (b) the type of T help necessary for B cells depends upon H-2-restricted contact between T and B cells, whereas, such contact-dependent help is not mandatory for the induction of virus-specific cytotoxic T cells.
Animals↗
Activation events during thymic selection.
During their differentiation in the mouse thymus, CD4+8- cells undergo several of the sequential changes observed upon normal activation of mature, peripheral CD4+ lymphocytes. Expression of CD69, an early activation marker, is first observed on a minority of cells at the T cell receptor (TCR)lo/med double-positive stage, is maximal (50-90%) on heat-stable antigen (HSA)hi TCRhi double-positive, HSAhi TCRmed CD4+8lo, and HSAhi TCRhi CD4+8- cells, and is downmodulated at the mature HSAlo CD4+8- stage. In contrast, CD44, a late activation marker, is selectively expressed at the HSAlo stage. The set of lymphokines that CD4+8- thymocytes can produce upon stimulation also characteristically expands from mainly interleukin 2 (IL-2) at the HSAhi stage, to IL-2 and very large amounts of IL-4, IL-5, IL-10, and interferon gamma (IFN-gamma) at the HSAlo stage. 1 in 30 HSAlo CD4+8- adult thymocytes secrete IL-4 upon stimulation through their TCR. This frequency is 25% of the frequency of IL-2 producers, about 100-fold above that of peripheral (mainly resting) CD4+ T cells. With time after their generation in organ culture, CD4+8- thymocytes lose their capacity to secrete IL-4, IL-5, and IFN-gamma, but not IL-2. Similarly, the frequency of IL-4, but not of IL-2, producers progressively decreases after emigration to the periphery as judged by direct comparison between thymic and splenic CD4+ cells in newborns, or by following the fate of intrathymically labeled CD4+8- cells in adults after their migration to the spleen. This sequence suggests that thymic selection results from an activation process rather than a simple rescue from death at the double-positive stage, and shows that the functional changes induced after intrathymic activation, although transient, are still evident after export to the periphery.
Animals↗
Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.
The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.
Animals↗
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.
Animals↗
The V beta 17+ T cell repertoire: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.
To ascertain how the actual repertoire of T cell receptors (TCRs) deviates from the theoretical, we have generated a large number of junctional region sequences from TCRs carrying the V beta 17 variable region. The greater than 600 sequences analyzed represent transcripts from nine different cell populations, permitting several comparisons: transcripts from an expressed vs. a non-expressed V beta 17 allele, those from E+ vs. E- mice, transcripts from immature vs. mature thymocytes, those from thymic vs. peripheral T cells, and those from CD4+ vs. CD8+ cells. These comparisons have allowed us to distinguish between the influence of molecular events involved in TCR gene rearrangement and that of various selection events that shape the T cell repertoire. Our most striking findings are: (a) that J beta usage is markedly skewed, partly due to recombination mechanics and partly due to selection forces: in particular, those mediated by the class II E molecule in the thymus; and (b) that TCRs on CD4+ and CD8+ cells show intriguing dissimilarities. In addition, we present evidence that N nucleotide additions occur with clear biases, probably due to idiosyncrasies of the recombination enzymes, and provide arguments that TCR and immunoglobulin CDR3s have distinct structures.
Alleles↗
Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.
The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.
Antibodies, Monoclonal↗