Αβ TCRs Research Articles

Immune surveillance of immune surveillance

The peptide repertoire presented by classical MHC Ia molecules is critical for immune surveillance of intracellular pathogens and cancer. The presentation of this normal peptide repertoire is regulated by ERAAP, the ER aminopeptidase associated with antigen processing. Changes in ERAAP function cause profound changes in the presented peptides that can potentially cause autoimmunity or alter the efficacy of immunotherapy of cancer. Thus, an unusual subset of CD8+ T cells monitors normal ERAAP function. Cells with compromised ERAAP activity present the QFL ligand that contains a highly conserved FL9 peptide presented by Qa-1b, a non-classical MHC Ib molecule expressed. The QFL-specific CD8+ T cells bear highly conserved αβ TCRs and have distinct anatomical locations as well as functions. Together these features implicate QFL-specific cells in carrying out immune surveillance of the MHC Ia immune surveillance pathway.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRβ and TCRα Genes and Influences Development of αβ T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRβ and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRβ and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αβ T cells, decreased genesis of invariant NK T lineage αβ T cells, and mature CD4+ αβ T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRβ and TCRα gene recombination and influence differentiation of αβ T lineage cells, thereby establishing replete diversity of αβ TCRs and populations of αβ T cells while restraining generation of potentially autoreactive conventional αβ T cells.

αβ T cells replacing dermal and epidermal γδ T cells in Tcrd-/- mice express an MHC-independent TCR repertoire.

The epidermis of mouse skin is usually populated by dendritic epidermal T cells (γδDETC) expressing an invariant Vγ5Vδ1+ TCR. In Tcrd-/- mice, skin-resident γδDETC are replaced by αβDETC carrying polyclonal αβ TCRs. Although they exhibit a dendritic morphology, αβDETC were reported to be less functional than genuine γδDETC, likely because their TCR is unable to interact with the original TCR ligands of γδDETC. However, the TCR repertoire of those replacement DETC in Tcrd-/- mice might provide clues for understanding the development and selection of canonical γδDETC. Here, we compare the phenotype and TCR repertoires of wild-type and Tcrd-/- mouse skin T cells. Our data reveal that αβDETC are CD4/CD8 double negative and express an MHC-independent TCR repertoire. Furthermore, we identify a second MHC-independent population of CD103hi CD4/ CD8 double-negative αβ T cells in the dermis of Tcrd-/- mice.

Unusual distribution of a non-classical MHC I restricted CD8+ T population

Abstract A broad repertoire of peptides is constitutively presented through the MHC I molecules on the cell surface. ER aminopeptidase associated with Ag processing (ERAAP), a critical molecule in the antigen processing and presentation pathway, has profound effects on the generation of this peptide repertoire. Interestingly, the ERAAP deficient cells can be effectively detected by a unique population of the cytotoxic CD8+ T cell, named QFL-T cells which are specific for the ligand presented by the non-classical MHC Ib molecule, Qa-1b. The QFL-T cells showed similar characteristics with the other non-classical MHC Ib restricted unconventional T cells, such as the NKT or MAIT cells in their semi-invariant αβ TCRs, but their function(s) remains unknown. To study the potential function of the QFL-T cells, we assessed a variety of tissues for the ligand expression and the presence of the QFL-T cells. We observed high expression of this ERAAP dependent Qa-1b restricted ligand and relatively high frequency of the QFL-T cells in the small intestine. Remarkably, the number and the frequency of QFL-T cells among the intraepithelial lymphocytes (IELs) was tightly associated with the age and the gut microbiota of the mice. These findings suggest a role for gut microbiota in the homing/maintenance of the QFL-T cells in the small intestine.

Structure of MHC-Independent TCRs and Their Recognition of Native Antigen CD155.

During normal T cell development in the thymus, αβ TCRs signal immature thymocytes to differentiate into mature T cells by binding to peptide-MHC ligands together with CD4/CD8 coreceptors. Conversely, in MHC and CD4/CD8 coreceptor-deficient mice, the thymus generates mature T cells expressing MHC-independent TCRs that recognize native conformational epitopes rather than linear antigenic-peptides presented by MHC. To date, no structural information of MHC-independent TCRs is available, and their structural recognition of non-MHC ligand remains unknown. To our knowledge in this study, we determined the first structures of two murine MHC-independent TCRs (A11 and B12A) that bind with high nanomolar affinities to mouse adhesion receptor CD155. Solution binding demonstrated the Vαβ-domain is responsible for MHC-independent B12A recognition of its ligand. Analysis of A11 and B12A sequences against various MHC-restricted and -independent TCR sequence repertoires showed that individual V-genes of A11 and B12A did not exhibit preference against MHC-restriction. Likewise, CDR3 alone did not discriminate against MHC binding, suggesting VDJ recombination together with Vα/Vβ pairing determine their MHC-independent specificity for CD155. The structures of A11 and B12A TCR are nearly identical to those of MHC-restricted TCR, including the conformations of CDR1 and 2. Mutational analysis, together with negative-staining electron microscopy images, showed that the CDR regions of A11 and B12A recognized epitopes on D1 domain of CD155, a region also involved in CD155 binding to poliovirus and Tactile in human. Taken together, MHC-independent TCRs adopt canonical TCR structures to recognize native Ags, highlighting the importance of thymic selection in determining TCR ligand specificity.

Coexpression of αβ and γδ TCRs bridges innate and adaptive immunity

T cells coexpressing αβ and γδ TCRs demonstrate characteristics of both αβ and γδ T cells, providing a link between innate and adaptive immune responses.

Unusual distribution of a non-classical MHC I restricted CD8+ T population

Abstract The normal peptide repertoire presented by MHC class I molecules on the cell surface is essential for immune surveillance of intracellular pathogens. The generation of this peptide repertoire is critically dependent upon the ER aminopeptidase associated with Ag processing (ERAAP). Interestingly, ERAAP-deficient cells are recognized by a unique population of cytotoxic T cells, called QFL-T cells, that are specific for the ligand presented by the non-classical MHC molecule, Qa-1b. The QFL-T cells are similar to other non-classical MHC restricted NKT or MAIT cells in their αβ TCRs, but their physiological function(s) are unknown. To identify the potential function of QFL-T cells, we assessed the expression of the Qa-1b restricted ligand as well as the distribution of QFL-T cells in various tissues. We found the ERAAP-dependent Qa-1b restricted ligand as well as the QFL-T cells were highly expressed in the intestine. Remarkably, relative to wild-type mice, the overall number and frequency of QFL-T cells among the intraepithelial lymphocytes(IELs) decreased significantly in the germ-free mice whereas the splenic QFL-T population remained unaffected. These findings suggest a role for gut-microbiota in the homing/maintenance of the QFL-T cells in the intestine.

MHC Restriction: Where Are We Now?

MHC Restriction: Where Are We Now?

A population of proinflammatory T cells coexpresses αβ and γδ T cell receptors in mice and humans

T cells are classically recognized as distinct subsets that express αβ or γδ TCRs. We identify a novel population of T cells that coexpress αβ and γδ TCRs in mice and humans. These hybrid αβ-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αβ TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1β and IL-23. Hybrid αβ-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.

Comparative analysis of CDR3 regions in paired human αβ CD8 T cells.

The majority of human CD8 cytotoxic T lymphocytes express αβ T‐cell receptors that recognize peptide–MHC class I complexes. Considerable attention has been devoted to TCR β repertoires, but study of TCR α chains has been limited. To gain a better understanding of the features of CDR3α and CDR3β in paired samples, we comprehensively analyzed 776 unique paired αβ TCR CDR3 regions in this study. We found that (I) the CDR3 length among paired αβ TCRs had a fairly narrow distribution due to random assortment of CDR3 length in alpha and beta chains; (II) nucleotide deletions among CDR3 regions were positively correlated with insertions in both α and β TCRs; (III) the CDR3 loops of both α and β chains contained an abundance of charged/polar residues and the CDR3 base regions contained a conserved motif; and (IV) the occurrence of Gly was CDR3 length‐ and position‐dependent in both chains, whereas the frequency of Ser at positions 106 and 107 was positively correlated with CDR3 length in TCR β. Overall, the amino acids in CDR3 loop regions were significantly different between TCR α and β, which suggests a distinct role for each chain in the recognition of antigen–MHC complexes. Here, we have provided detailed information on CDR3 in paired TCRs expressed on human CD8+ T cells and established the basis of a reference set for αβ TCR repertoires in healthy humans.

Previously Hidden Dynamics at the TCR–Peptide–MHC Interface Revealed

A structural characterization of the interaction between αβ TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.

Invariant NKT Cells and Control of the Thymus Medulla.

Most αβ T cells that form in the thymus are generated during mainstream conventional thymocyte development and involve the generation and selection of a diverse αβ TCR repertoire that recognizes self-peptide/MHC complexes. Additionally, the thymus also supports the production of T cell subsets that express αβ TCRs but display unique developmental and functional features distinct from conventional αβ T cells. These include multiple lineages of CD1d-restricted invariant NKT (iNKT) cells that express an invariant αβ TCR, branch off from mainstream thymocytes at the CD4+CD8+ stage, and are potent producers of polarizing cytokines. Importantly, and despite their differences, iNKT cells and conventional αβ T cells share common requirements for thymic epithelial microenvironments during their development. Moreover, emerging evidence suggests that constitutive cytokine production by iNKT cells influences both conventional thymocyte development and the intrathymic formation of additional innate CD8+ αβ T cells with memory-like properties. In this article, we review evidence for an intrathymic innate lymphocyte network in which iNKT cells play key roles in multiple aspects of thymus function.

Self-reactive T cells restricted by non-classical MHC Ib are associated with metabolic disease

Abstract The normal peptide repertoire presented by classical and non-classical MHC class I molecules is regulated by ERAAP, the ER aminopeptidase associated with antigen processing. Loss of ERAAP’s peptide trimming function in cells causes dramatic changes in the peptide repertoire. The changes in the peptide repertoire enhance the immunogenicity of ERAAP-deficient cells and elicit potent immune responses in otherwise syngeneic wild-type mice. Normal ERAAP function is monitored by an unusual subset of semi-invariant CD8+ T cells that recognize the QFL ligand. The QFL ligand consists of a peptide encoded by the highly conserved FAM49a/b genes that is presented by Qa-1b, a non-classical MHC Ib molecule only on surface of ERAAP-deficient cells. We show that these QFL-specific CD8+ T cells (QFL-T cells) bear unique and semi-invariant αβ TCRs. Genetic manipulation of the expression of the self-QFL ligand and functional characteristics of QFL-T cells shows that in addition to monitoring ERAAP function, QFL-T cells may also regulate metabolic activity.

Antigen Processing in the Endoplasmic Reticulum Is Monitored by Semi-Invariant αβ TCRs Specific for a Conserved Peptide-Qa-1b MHC Class Ib Ligand.

Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide-MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I-presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αβ TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRβ-transgenic mice were assessed for QFL-specific TCR α- and β-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3-TRAJ21 α-chain and TRBV5-TRBD1-TRBJ2-7 β-chain gene segments. Furthermore, the CDR3 regions of the α- as well as β-chains were required for QFL ligand recognition. Thus, the αβ TCRs used to recognize the peptide-Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.

Innate PLZF+CD4+ αβ T cells develop and expand in the absence of Itk.

T cell development in the thymus produces multiple lineages of cells, including innate T cells. Studies in mice harboring alterations in TCR signaling proteins or transcriptional regulators have revealed an expanded population of CD4(+) innate T cells in the thymus that produce IL-4 and express the transcription factor promyelocytic leukemia zinc finger (PLZF). In these mice, IL-4 produced by the CD4(+)PLZF(+) T cell population leads to the conversion of conventional CD8(+) thymocytes into innate CD8(+) T cells resembling memory T cells expressing eomesodermin. The expression of PLZF, the signature invariant NKT cell transcription factor, in these innate CD4(+) T cells suggests that they might be a subset of αβ or γδ TCR(+) NKT cells or mucosal-associated invariant T (MAIT) cells. To address these possibilities, we characterized the CD4(+)PLZF(+) innate T cells in itk(-/-) mice. We show that itk(-/-) innate PLZF(+)CD4(+) T cells are not CD1d-dependent NKT cells, MR1-dependent MAIT cells, or γδ T cells. Furthermore, although the itk(-/-) innate PLZF(+)CD4(+) T cells express αβ TCRs, neither β2-microglobulin-dependent MHC class I nor any MHC class II molecules are required for their development. In contrast to invariant NKT cells and MAIT cells, this population has a highly diverse TCRα-chain repertoire. Analysis of peripheral tissues indicates that itk(-/-) innate PLZF(+)CD4(+) T cells preferentially home to spleen and mesenteric lymph nodes owing to increased expression of gut-homing receptors, and that their expansion is regulated by commensal gut flora. These data support the conclusion that itk(-/-) innate PLZF(+)CD4(+) T cells are a novel subset of innate T cells.

IL-2: the first effective immunotherapy for human cancer.

The ability of IL-2 to expand T cells with maintenance of functional activity has been translated into the first reproducible effective human cancer immunotherapies. The administration of IL-2 can lead to durable, complete, and apparently curative regressions in patients with metastatic melanoma and renal cancer. The growth of large numbers of tumor-infiltrating lymphocytes with in vitro anti-cancer activity in IL-2 has led to the development of cell transfer therapies that are highly effective in patients with melanoma. The genetic modification of T cells with genes encoding αβ TCRs or chimeric Ag receptors and the administration of these cells after expansion in IL-2 have extended effective cell transfer therapy to other cancer types.

Check MAIT.

The evolutionary conservation of T lymphocyte subsets bearing αβ TCRs using invariant α-chains is indicative of unique and important functions. Among these T lymphocytes, NKT cells that express an invariant TCRα-chain and recognize lipid Ags presented by the nonclassical MHC class I molecule CD1d are probably the most studied. However, a new population of evolutionarily conserved T cells with another invariant TCRα rearrangement was recently characterized. These cells, which are very abundant in humans, tend to reside in mucosal tissues and, therefore, were named mucosal-associated invariant T (MAIT) cells. Until recently, little was known about MAIT cells; however, several recent advances in our understanding of MAIT cell characteristics and functions secure their upcoming rise to fame in the immunology field and in clinical practice.

Peripheral CD4CD8 Double Positive T Cells with a Distinct Helper Cytokine Profile Are Increased in Rheumatoid Arthritis

Peripheral CD4CD8 double positive (DP) T cells have been reported to play a role in several autoimmune diseases, virus infections and cancer. In rheumatoid arthritis (RA), both CD4 and CD8 single positive (SP) T cells are known to be involved in the pathogenesis, but the role of peripheral CD4CD8 DP T cells has not been investigated in detail. Anti cyclic citrullinated antibodies (ACPA) positive and ACPA negative RA patients, patients with systemic lupus erythematodes (SLE) and age matched healthy donors (HD) were enrolled in the analysis. The frequencies and phenotype of DP T cells in PBMC were investigated. In addition, DP T cells were quantified in biopsies from rheumatoid synovium. After in vitro restimulation, the cytokine production of DP T cells was investigated in cultures of PBMC. CMV specific cytokine secretion as well as proliferation was analyzed following antigen specific restimulation after an appropriate culture duration. DP T cells were found more frequently in RA patients than in healthy controls or patients with SLE. These DP T cells express αβ TCRs, are of a memory phenotype and share features of both CD4 as well as CD8 SP T cells. Importantly, DP T cells were found to also be present in the rheumatoid synovium. Further characterization of DP T cells from RA patients revealed increased production of IL-21 and IL-4, implying a possible role as T helper cells. In addition, DP T cells in RA seem to contribute to the inflammatory process, because they produce significantly more IFNγ than counterparts from HD and are increased in CMV+ RA patients. Given their capacity to produce a variety of cytokines (IL4, IL21 and IFNγ), their association with ACPA positive RA and their presence in the synovium, we suggest an important role of double positive T cells in the pathogenesis of rheumatoid arthritis.

A leaky mutation in CD3D differentially affects αβ and γδ T cells and leads to a Tαβ–Tγδ+B+NK+ human SCID

T cells recognize antigens via their cell surface TCR and are classified as either αβ or γδ depending on the variable chains in their TCR, α and β or γ and δ, respectively. Both αβ and γδ TCRs also contain several invariant chains, including CD3δ, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3δ-deficient patients described to date showed a complete block in T cell development. However, CD3δ-KO mice have an αβ T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in αβ but not in γδ T cell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3δ proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant αβ T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of αβ T cells, surface TCR expression was more reduced in γδ than in αβ T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3δ requirements for αβ versus γδ T cell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.

Vγ2Vδ2 T Cell Receptor Recognition of Prenyl Pyrophosphates Is Dependent on All CDRs

Abstract γδ T cells differ from αβ T cells in the Ags they recognize and their functions in immunity. Although most αβ TCRs recognize peptides presented by MHC class I or II, human γδ T cells expressing Vγ2Vδ2 TCRs recognize nonpeptide prenyl pyrophosphates. To define the molecular basis for this recognition, the effect of mutations in the TCR CDR was assessed. Mutations in all CDR loops altered recognition and cover a large footprint. Unlike murine γδ TCR recognition of the MHC class Ib T22 protein, there was no CDR3δ motif required for recognition because only one residue is required. Instead, the length and sequence of CDR3γ was key. Although a prenyl pyrophosphate-binding site was defined by Lys109 in Jγ1.2 and Arg51 in CDR2δ, the area outlined by critical mutations is much larger. These results show that prenyl pyrophosphate recognition is primarily by germline-encoded regions of the γδ TCR, allowing a high proportion of Vγ2Vδ2 TCRs to respond. This underscores its parallels to innate immune receptors. Our results also provide strong evidence for the existence of an Ag-presenting molecule for prenyl pyrophosphates.