How the T cell repertoire becomes peptide and MHC specific

T cells bearing alphabeta T cell receptors (TCRs) recognize antigens in the form of peptides bound to class I or class II major histocompatibility proteins (MHC). TCRs on mature T cells are usually very specific for both peptide and MHC class and allele. They are picked out from a precursor population in the thymus by MHC-driven positive and negative selection. Here we show that the pool of T cells initially positively selected in the thymus contains many T cells that are very crossreactive for peptide and MHC and that subsequent negative selection establishes the MHC-restriction and peptide specificity of peripheral T cells. Our results also suggest that germline-encoded TCR variable elements have an inherent predisposition to react with features shared by all MHC proteins.     

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.     

Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection

The failure to eliminate self-reactive T cells during negative selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T-cell receptors (TCRs) may form unstable complexes with self-peptide-MHC by adopting suboptimal binding topologies compared with anti-microbial TCRs. Alternatively, escape can occur by weak binding between self-peptides and MHC. We determined the structure of a human autoimmune TCR (MS2-3C8) bound to a self-peptide from myelin basic protein (MBP) and the multiple sclerosis-associated MHC molecule HLA-DR4. MBP is loosely accommodated in the HLA-DR4-binding groove, accounting for its low affinity. Conversely, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti-microbial TCRs. MS2-3C8 engages self-antigen via a docking mode that resembles the optimal topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Combined with a unique CDR3β conformation, this docking mode compensates for the weak binding of MBP to HLA-DR4 by maximizing interactions between MS2-3C8 and MBP. Thus, the MS2-3C8-MBP-DR4 complex reveals the basis for an alternative strategy whereby autoreactive T cells escape negative selection, yet retain the ability to initiate autoimmunity.     

Normal T Cell Selection Occurs in CD205-Deficient Thymic Microenvironments

The thymus imparts a developmental imprint upon T cells, screening beneficial and self-tolerant T cell receptor (TCR) specificities. Cortical thymic epithelial cells (CTEC) present self-peptide self-MHC complexes to thymocytes, positively selecting those with functional TCRs. Importantly, CTEC generate diverse self-peptides through highly specific peptide processing. The array of peptides utilized for positive selection appears to play a key role in shaping TCR repertoire and influencing T cell functionality. Whilst self-peptide diversity influences T cell development, the precise source of proteins generating such self-peptide arrays remains unknown, the abundance of apoptotic thymocytes failing thymic selection may provide such a pool of self-proteins. In relation to this notion, whilst it has been previously demonstrated that CTEC expression of the endocytic receptor CD205 facilitates binding and uptake of apoptotic thymocytes, the possible role of CD205 during intrathymic T cell development has not been studied. Here, we directly address the role of CD205 in normal thymocyte development and selection. Through analysis of both polyclonal and monoclonal transgenic TCR T-cell development in the context of CD205 deficiency, we demonstrate that CD205 does not play an overt role in T cell development or selection.     

Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes.

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.     

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

The mammalian alpha/beta T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR-MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential.     

Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection

Thymic selection is controlled in part by the avidity of the interaction between thymocytes and APCs. In agreement, the selective outcome can be modulated by altering the expression levels of selecting ligands on APCs. Here we test the converse proposition, i. e., whether changing TCR levels on thymocytes can alter the selective outcome. To this end, we have generated mice in which all thymocytes express two transgenic TCRs simultaneously (dual TCR-expressing (DTE) mice), the class I-restricted HY TCR and the class II-restricted AND TCR. Due to mutual dilution, surface expression levels of the two individual transgenic TCRs are diminished in DTE relative to single TCR-expressing mice. We find that thymic selection is highly sensitive to these reductions in TCR surface expression. Positive selection mediated by the AND and HY TCRs is severely impaired or abolished, respectively. Negative selection of the HY TCR in male DTE mice is also partly blocked, leading to the appearance of significant numbers of double positive thymocytes. Also, in the periphery of male, but not female, DTE mice, substantial numbers of single positive CD8bright cells accumulate, which are positively selected in the thymus but by a highly inefficient hemopoietic cell-dependent process. Overall our results favor the interpretation that the outcome of thymic selection is not determined solely by avidity and the resulting signal intensity, but is also constrained by other factors such as the nature of the ligand and/or its presentation by different subsets of APCs.     

Deriving quantitative constraints on T cell selection from data on the mature T cell repertoire

The T cell repertoire is shaped in the thymus through positive and negative selection. Thus, data about the mature repertoire may be used to infer information on how TCR generation and selection operate. Assuming that T cell selection is affinity driven, we derive the quantitative constraints that the parameters driving these processes must fulfill to account for the experimentally observed levels of alloreactivity, self MHC restriction and the frequency of cells recognizing a given foreign Ag. We find that affinity-driven selection is compatible with experimental estimates of these latter quantities only if 1) TCRs see more peptide residues than MHC polymorphic residues, 2) the majority of positively selected clones are deleted by negative selection, 3) between 1 and 3.6 clonal divisions occur on average in the thymus after completion of TCR rearrangement, and 4) selection is driven by 103-105 self peptides.     

Opposite effects of endogenous peptide-MHC class I on T cell activity in the presence and absence of CD8

Nonstimulatory or endogenous peptide-MHC (pepMHC) presented on the surfaces of APCs, either alone or alongside agonist pepMHC, plays various roles in T cell selection and activation. To examine these properties in more detail, we explored several model systems of TCR and pepMHC ligands with sufficient affinity to be activated in the absence of CD8. The TCRs had a range of affinities for agonist and nonstimulatory ligands and were restricted by MHC class I alleles with different properties. We observed CD8-independent antagonism from TCR-pepMHC interactions with very low affinities (e.g., K(D) = 300 μM). In addition, endogenous peptide-L(d) complexes on APCs antagonized activation of coreceptor (CD8)-negative 2C T cells even by the strong agonist QL9-L(d). In contrast, TCRs m33 and 3D-PYY, restricted by K(b) and D(b), respectively, did not show signs of antagonism by endogenous pepMHC in the absence of CD8. This did not appear to be an inherent difference in the ability of the TCRs to be antagonized, as altered peptide ligands could antagonize each TCR. In the presence of CD8, endogenous pepMHC ligands acted in some cases as coagonists. These results show that endogenous pepMHC molecules exhibit complex behavior in T cells, leading to either reduced activity (e.g., in cases of low coreceptor levels) or enhanced activity (e.g., in presence of coreceptor). The behavior may be influenced by the ability of different TCRs to recognize endogenous pepMHC but also perhaps by the inherent properties of the presenting MHC allele.     

Alpha beta TCRs differ in the degree of their specificity for the positively selecting MHC/peptide ligand

We have tested the peptide specificity of positive selection using three transgenic alphabetaTCRs, originally selected on class II MHC (A(b)) covalently bound with one peptide Ealpha (52-68) (Ep). The transgenic TCR specific for the cytochrome c-derived (43-58) peptide was selected on A(b) bound with different arrays of endogenous peptides or the analogue of Ep covalently bound to A(b), but not on the original A(b)Ep complex. In contrast, transgenic TCRs specific for two different analogues of the Ep peptide and A(b) did not mature as CD4(+) T cells in various thymic environments, including the A(b)EpIi(-) mice. These results show that TCRs can be promiscuous or specific for the selecting MHC/peptide complex, and suggest that in mice described in this study transgenic expression of the TCR changes the original requirements for the positively selecting MHC/peptide complex. Future studies will determine whether the latter phenomenon is general or specific for this system.     

Physical and functional bivalency observed among TCR/CD3 complexes isolated from primary T cells

Unlike BCR and secreted Ig, TCR expression is not thought to occur in a bivalent form. The conventional monovalent model of TCR/CD3 is supported by published studies of complexes solubilized in the detergent digitonin, in which bivalency was not observed. We revisited the issue of TCR valency by examining complexes isolated from primary αβ T cells after solubilization in digitonin. Using immunoprecipitation followed by flow cytometry, we unexpectedly observed TCR/CD3 complexes that contained two TCRs per complex. Standard anti-TCR Abs, being bivalent themselves, tended to bind with double occupancy to bivalent TCRs; this property masked the presence of the second TCR per complex in certain Ab binding assays, which may partially explain why previous data did not reveal these bivalent complexes. We also found that the prevalence of bivalency among fully assembled, mature TCR/CD3 complexes was sufficient to impact the functional performance of immunoprecipitated TCRs in binding antigenic peptide/MHC-Ig fusion proteins. Both TCR positions per bivalent complex required an Ag-specific TCR to effect optimal binding to these soluble ligands. Therefore, we conclude that in primary T cells, TCR/CD3 complexes can be found that are physically and functionally bivalent. The expression of bivalent TCR/CD3 complexes has implications regarding potential mechanisms by which Ag may trigger signaling. It also suggests the possibility that the potential for bivalent expression could represent a general feature of Ag receptors.     

Population variability in the generation and selection of T-cell repertoires

The diversity of T-cell receptor (TCR) repertoires is achieved by a combination of two intrinsically stochastic steps: random receptor generation by VDJ recombination, and selection based on the recognition of random self-peptides presented on the major histocompatibility complex. These processes lead to a large receptor variability within and between individuals. However, the characterization of the variability is hampered by the limited size of the sampled repertoires. We introduce a new software tool SONIA to facilitate inference of individual-specific computational models for the generation and selection of the TCR beta chain (TRB) from sequenced repertoires of 651 individuals, separating and quantifying the variability of the two processes of generation and selection in the population. We find not only that most of the variability is driven by the VDJ generation process, but there is a large degree of consistency between individuals with the inter-individual variance of repertoires being about ∼2% of the intra-individual variance. Known viral-specific TCRs follow the same generation and selection statistics as all TCRs.     

The T cell receptor's alpha-chain connecting peptide motif promotes close approximation of the CD8 coreceptor allowing efficient signal initiation

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The alpha-chain connecting peptide motif (alpha-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR alpha-chain. TCRs lacking the alpha-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant alpha-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the alpha-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the alpha-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.     

Dendritic cells sensitize TCRs through self-MHC-mediated Src family kinase activation

It is unclear whether peptide-MHC class II (pMHC) complexes on distinct types of APCs differ in their capacity to trigger TCRs. In this study, we show that individual cognate pMHC complexes displayed by dendritic cells (DCs), as compared with nonprofessional APCs, are far better in productively triggering Ag-specific TCRs independently of conventional costimulation. As we further show, this is accomplished by the unique ability of DCs to robustly activate the Src family kinases (SFKs) Lck and Fyn in T cells even in the absence of cognate peptide. Instead, this form of SFK activation depends on interactions of DC-displayed MHC with TCRs of appropriate restriction, suggesting a central role of self-pMHC recognition. DC-mediated SFK activation leads to "TCR licensing," a process that dramatically increases sensitivity and magnitude of the TCR response to cognate pMHC. Thus, TCR licensing, besides costimulation, is a main mechanism of DCs to present Ag effectively.     

Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor.

Using NMR spectroscopy, we determined the solution structure of a single-chain T-cell receptor (scTCR) derived from the major histocompatibility complex (MHC) class II-restricted D10 TCR. The conformations of complementarity-determining regions (CDRs) 3beta and 1alpha and surface properties of 2alpha are different from those of related class I-restricted TCRs. We infer a conserved orientation for TCR V(alpha) domains in complexes with both class I and II MHC-peptide ligands, which implies that small structural variations in V(alpha) confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond time scale) provides insight into immune recognition and selection mechanisms.     

T-cell receptor structure and TCR complexes

The first crystal structures of intact T-cell receptors (TCRs) and their complexes with MHC peptide antigens (pMHC) were reported during the past year, along with those of a single-chain TCR Fv fragment and a β-chain complexed with two different bacterial superantigens. These structures have shown the similarities and differences in the architecture of the antigen-binding regions of TCRs and antibodies, and how the TCR interacts with pMHC ligands as well as with superantigens     

A role for the alpha-chain connecting peptide motif in mediating TCR-CD8 cooperation

To generate peripheral T cells that are both self-MHC restricted and self-MHC tolerant, thymocytes are subjected to positive and negative selection. How the TCR discriminates between positive and negative selection ligands is not well understood, although there is substantial evidence that the CD4 and CD8 coreceptors play an important role in this cell fate decision. We have previously identified an evolutionarily conserved motif in the TCR, the alpha-chain connecting peptide motif (alpha-CPM), which allows the TCR to deliver positive selection signals. Thymocytes expressing alpha-CPM-deficient receptors do not undergo positive selection, whereas their negative selection is not impaired. In this work we studied the ligand binding and receptor function of alpha-CPM-deficient TCRs by generating T cell hybridomas expressing wild-type or alpha-CPM-deficient forms of the T1 TCR. This K(d)-restricted TCR is specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide(252-260) IASA-YIPSAEK(ABA)I and is therefore amenable to TCR photoaffinity labeling. The experiments presented in this work show that alpha-CPM-deficient TCRs fail to cooperate with CD8 to enhance ligand binding and functional responses.     

Structure-activity relationship of T-cell receptors based on alanine scanning

T-cell receptors (TCR) recognize complexes between human leukocyte antigens (HLA) and peptides derived from intracellular proteins. Their therapeutic use for antigen targeting, however, has been hindered by the very low binding affinity of TCRs, typically in the 1- to 100-μM range. Therefore, to construct mutant TCRs with high binding affinity, we need to understand the relationship between the structure and activity of these molecules. Here, we attempted to identify the amino acids of the TCR that are important for binding to the peptide/HLA complex. We used a TCR that recognizes complexes between HLA-A^∗0201 and the peptide from tyrosinase, antigen overexpressed in melanoma. We changed 16 amino acids in the third complementarity-determining region within the TCR to alanine and examined the effect on binding affinity. Five alanine substitutions decreased the binding affinity to below 10% compared with that of wild-type TCR. In contrast, one alanine substitution caused a faster on-rate and slower off-rate, and increased the binding affinity to three times that of the wild-type TCR. Our results provide fundamental information for constructing mutant TCRs with high binding affinity.     

Directed evolution of human T-cell receptors with picomolar affinities by phage display

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide-human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to approximately 1-100 muM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax(11-19) peptide-HLA-A(*)0201 complex and the NY-ESO-1(157-165) tumor-associated peptide antigen-HLA-A(*)0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.     

Extrathymic selection of TCR gamma delta + T cells by class II major histocompatibility complex molecules

The influence of MHC antigens on TCR gamma delta usage in CD8+ intraepithelial lymphocytes (IELs) was examined using a pan-reactive and V delta 4 region-specific MAb. While an average of 30% of IELs from the majority of mice of various MHC haplotypes were V delta 4+, a 2-fold or greater percentage of IELs from H-2k mice were V delta 4+. Analysis of IELs from F1 mice indicated that the increase in TCRs using V delta 4 was likely to be the result of positive selection. The V delta 4 usage patterns of IELs from recombinant inbred strains and from mice recombinant within H-2 revealed that the increase in V delta 4 usage mapped to H-2 and required I-E expression. Moreover, selection of TCRs using V delta 4 occurred in chimeric mice in the absence of a thymus. The results demonstrate an extrathymic selective mechanism for gamma delta TCRs of CD8+ IELs and suggest that these cells may exhibit MHC class II-restricted antigen recognition.