Class II-restricted T-cell Receptor Research Articles

High-throughput discovery of MHC class I- and II-restricted T cell epitopes using synthetic cellular circuits.

Antigen discovery technologies have largely focused on major histocompatibility complex (MHC) class I-restricted human T cell receptors (TCRs), leaving methods for MHC class II-restricted and mouse TCR reactivities relatively undeveloped. Here we present TCR mapping of antigenic peptides (TCR-MAP), an antigen discovery method that uses a synthetic TCR-stimulated circuit in immortalized T cells to activate sortase-mediated tagging of engineered antigen-presenting cells (APCs) expressing processed peptides on MHCs. Live, tagged APCs can be directly purified for deconvolution by sequencing, enabling TCRs with unknown specificity to be queried against barcoded peptide libraries in a pooled screening context. TCR-MAP accurately captures self-reactivities or viral reactivities with high throughput and sensitivity for both MHC class I-restricted and class II-restricted TCRs. We elucidate problematic cross-reactivities of clinical TCRs targeting the cancer/testis melanoma-associated antigen A3 and discover targets of myocarditis-inciting autoreactive T cells in mice. TCR-MAP has the potential to accelerate T cell antigen discovery efforts in the context of cancer, infectious disease and autoimmunity.

Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors

CD4+ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC50] <10 nM), or C61 and preS9 (EC50 <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4+ and CD8+ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4+ T cell help in viral infection with potential benefit for T cell therapy.

OS12.4.A MHC class II-restricted transgenic T cell receptor therapy targeting mutant capicua transcriptional repressor in experimental gliomas

Abstract BACKGROUND Glioma subtypes are classified according to their characteristic mutations and show a high degree of resistance to standard therapeutic interventions such as radiotherapy and alkylating chemotherapy. Some of these characteristic mutations have shown to generate immunogenic neoepitopes that can be targeted with immunotherapy. 70% of oligodendrogliomas carry capicua transcriptional repressor (CIC) inactivating mutations. RESULTS In a screen for potential immunogenic glioma neoepitopes we identified recurrent CIC hotspot mutations at position 215 (CICR215W/Q) expressed in a subset of oligodendrogliomas as an immunogenic major histocompatibility complex (MHC) class II-restricted neoepitopes. Peptide-based vaccination of MHC-humanized mice resulted in the generation of robust mutation-specific T cell responses against CICR215W/Q, restricted to MHC class II. Droplet-based single cell T cell receptor (TCR) sequencing from CICR215W-specific T cell lines enabled retrieval of MHC class II-restricted CICR215W-reactive TCRs. By retroviral transduction of T cells, we established a flow cytometry-based testing platform of retrieved TCRs and were able to show the top reactive TCR against CICR215W to be shared between individual mice. Using a newly developed glioma model in MHC-humanized mice induced by CRISPR-based delivery of tumor suppressor targeting guide RNAs, we show that adoptive intraventricular transfer of CICR215W-specific TCR-transgenic T cells exert anti-tumor responses against CICR215W-expressing syngeneic gliomas. CONCLUSION The integration of immunocompetent MHC-humanized orthotopic glioma models in the discovery of shared immunogenic glioma neoepitopes facilitates the identification and preclinical testing of HLA-restricted neoepitope-specific TCRs for locoregional TCR-transgenic T cell adoptive therapy.

OS06.4A Identification of T cell receptors targeting ZFTA-RELA fusion-positive ependymoma

Abstract BACKGROUND The oncogenic gene fusion between ZFTA (formerly C11orf95) and RELA has in recent years been highlighted as oncogenic driver event ultimately leading to malignant transformation and progression of ependymoma. Representing a genetic hallmark in 17.6% of ependymomas, ZFTA-RELA fusion-positive tumors qualified as separate diagnostic entity in the 2016 revision of the WHO classification of CNS tumors. This tumor entity mainly occurs in pediatric patients and is characterized by poor prognosis and the lack of biology-driven therapy. RESULTS De novo prediction of putative gene fusion-derived neoepitopes in malignant CNS diseases yielded several potential candidates suitable for screening. Of these, vaccination of A2.DR1-transgenic major histocompatibility complex (MHC)-humanized mice with in silico compiled peptide vaccines encompassing the ZFTA-RELA fusion sequence elicited robust antigen-specific CD4+-restricted immune responses. We identified ZFTA-RELA-reactive T cell clones by multiplexed Interferon-γ / Interleukin-2 recall response. Single-cell VDJ-sequencing of reactive T cells demonstrated a highly clonal T cell receptor (TCR) repertoire and shared clonotypes among all vaccinated animals. Matching TCRα/β chains have been assembled from single-cell sequencing data and cloned for functional testing of neoepitope-reactive TCR-transgenic T cells in vitro as well as in vivo using a transposon system-based immunocompetent, MHC-humanized ZFTA-RELA fusion-positive ependymoma model. CONCLUSION Here we identify the oncogenic ZFTA-RELA gene fusion product as a novel tumor-specific neoepitope that is recognized by MHC class II-restricted TCRs. Therapeutic efficacy of TCR-transgenic cell therapy can be further investigated using an immunocompetent MHC-humanized mouse model of ZFTA-RELA fusion-positive ependymoma. Our findings provide initial evidence for neoepitope-specific immunotherapy in pediatric CNS tumors.

Targeting KRAS mutations with HLA class II-restricted TCRs for the treatment of solid tumors

ABSTRACT T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.

Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy

T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4+ and CD8+ T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT+ melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.

Peptide–MHC Binding Reveals Conserved Allosteric Sites in MHC Class I- and Class II-Restricted T Cell Receptors (TCRs)

T cells are vital for adaptive immune responses that protect against pathogens and cancers. The T cell receptor (TCR)–CD3 complex comprises a diverse αβ TCR heterodimer in noncovalent association with three invariant CD3 dimers. The TCR is responsible for recognizing antigenic peptides bound to MHC molecules (pMHC), while the CD3 dimers relay activation signals to the T cell. However, the mechanisms by which TCR engagement by pMHC is transmitted to CD3 remain mysterious, although there is growing evidence that mechanosensing and allostery both play a role. Here, we carried out NMR analysis of a human autoimmune TCR (MS2-3C8) that recognizes a self-peptide from myelin basic protein presented by the MHC class II molecule HLA-DR4. We observed pMHC-induced NMR signal perturbations in MS2-3C8 that indicate long-range effects on TCR β chain conformation and dynamics. Our results demonstrate that, in addition to expected changes in the NMR resonances of pMHC-contacting residues, perturbations extend to the Vβ/Vα, Vβ/Cβ, and Cβ/Cα interfacial regions. Moreover, the pattern of long-range perturbations is similar to that detected previously in the β chains of two MHC class I-restricted TCRs, thereby revealing a common allosteric pathway among three unrelated TCRs. Molecular dynamics (MD) simulations predict similar pMHC-induced effects. Taken together, our results demonstrate that pMHC binding induces long-range allosteric changes in the TCR β chain at conserved sites in both representative MHC class I- and class II-restricted TCRs, and that these sites may play a role in the transmission of signaling information.

Characterization of MHC class II-restricted T-cell receptors for T-cell therapy of HBV infection

Background & Aim Hepatitis B virus (HBV) infection remains a severe health problem with current treatment options being unable to achieve viral clearance. Since virus eradication is known to be accompanied by a strong T-cell response in patients with resolved infection, adoptive T-cell therapy represents a promising therapeutic approach. We recently demonstrated that CD8+ T cells grafted with T-cell receptors (TCR) restricted by MHC class I have the potential to cure HBV infection in vitro and in vivo. Nevertheless, also CD4+ T cells are known to play an important role in resolving HBV infection and may therefore potentially benefit T-cell therapy. We thus identified, cloned and characterized MHC class II-restricted TCRs from HBV-specific CD4+ T cells. Methods, Results & Conclusion HBV peptides were used to stimulate PBMC from donors with cleared HBV infection. HBV-specific CD4+ T cells secreting TNF-α were sorted by flow cytometry and clonally expanded. Subsequently, variable sequences of TCR α- and β-chains were identified, codon-optimized and cloned into a retroviral vector with murine constant domains. After retroviral transduction, all TCRs were expressed on primary T cells and characterized in depth regarding their MHC restriction, specificity, binding affinity and functionality. A total of 23 TCRs specific for 7 different epitopes from HBV envelope, core or polymerase protein were generated. Through retroviral titration, TCR integration was limited to under 5 integrates per cell, as determined by qPCR. Transduction rates ranging from 65-85% were measured by flow cytometry, where strength of surface expression was found to be characteristic of the TCR rather than the number of integrates. Through co-culture with partially matched lymphoblastoid cell lines, 6 different MHC class II restrictions were identified, including HLA-DRB1*01, *07, *04 and *13 or HLA-DPB1*02 and *04. 17 TCRs recognized at least 3 HBV genotype variants and 12 TCRs had a high binding affinity with EC50 values in a low nanomolar range. Similarly, 12 TCRs were able to recognize endocytosed and intracellularly processed viral antigen. 20 TCRs were capable of activating CD8+ T cells, inducing granzyme B secretion in 75-100% of transduced T cells. Taken together, our set of 23 MHC class II-restricted TCRs recognizes epitopes derived from HBV core, surface and polymerase protein. These TCRs enabled both CD4+ and CD8+ T cells to detect antigen on MHC class II in a nanomolar range, resulting in antigen-specific T-cell activation.

Abstract CT003: A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer

Abstract Background: Adoptive transfer of genetically-modified T cells is being explored as a salvage treatment for patients with selected metastatic cancers. Most of the current strategies utilize MHC class I-restricted T cell receptor (TCR) or chimeric antigen receptor (CAR) technologies to genetically modify CD8+ T cells or bulk T cells for patient treatment. Evidence indicates that CD4+ T cells can induce tumor regression, similar to CD8+ T cells. To test this hypothesis, an HLA-DPB1*0401-restricted TCR recognizing MAGE-A3 was isolated from a patient's peripheral blood after MAGE-A3 peptide vaccination. Because HLA-DPB1*0401 is present in ∼60% of the Caucasian population and MAGE-A3 is expressed in up to one third of tumor specimens from a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients. Trial Design: Eligible patients were HLA-DPB1*0401 positive with MAGE-A3 positive tumor specimens, and had not responded or had recurred following at least one standard first line therapy for their disease. Patients received a lymphodepleting preparative regimen, followed by adoptive transfer of purified CD4+ T cells transduced with the HLA-DPB1*0401-restricted MAGE-A3 TCR plus systemic high-dose interleukin-2 (IL-2). A cell dose-escalation was conducted, treating 1 patient at each cohort (0.01, 0.03, 0.1, up to 30 billion cells), followed by 6 patients at the highest dose level (up to 100 billion cells). Clinical trial information: NCT02111850. Results: Fourteen patients were treated in this phase I study, including the last 6 patients at the highest dose level (78∼100 billion cells). Objective partial responses (RECIST) were observed in a patient with metastatic cervical cancer (ongoing at 11+ months), esophageal cancer (duration 3 months) and urothelial cancer (ongoing at 4+ months). High levels of IL-6 were detected in all patients’ serum samples after adoptive cell transfer. One month after the treatment, TCR-transduced T cells persisted at high levels in the peripheral blood of 6 patients who received the highest dose level, compared to patients who receive lower dose levels (0.01 ∼ 30 billion cells) (p = 0.0082). These results demonstrate the safety of administering autologous CD4+ T cells genetically-engineered to express an MHC class II-restricted anti-tumor TCR targeting MAGE-A3 and presents evidence for efficacy. We have started the phase II clinical trial to study the efficacy of this TCR therapy in different types of metastatic cancer. This clinical trial extends the reach of TCR gene therapy to patients with metastatic cancer. To our knowledge, this clinical trial represents the first genetically-modified CD4+ T cell immunotherapy against metastatic cancer. Citation Format: Yong-Chen Lu, Linda L. Parker, Tangying Lu, Zhili Zheng, Xin Yao, Paul F. Robbins, Steven A. Feldman, Pierre van der Bruggen, Christopher A. Klebanoff, Christian S. Hinrichs, Stephanie L. Goff, Richard M. Sherry, Udai S. Kammula, James C. Yang, Steven A. Rosenberg. A phase I study of an HLA-DPB1*0401-restricted T-cell receptor targeting MAGE-A3 for patients with metastatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT003.

Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.

The atypical MAPK ERK3 controls positive selection of thymocytes.

Extracellular signal-regulated kinase 3 (ERK3 )is an atypical member of the mitogen-activated protein kinase (MAPK) family. We have previously shown that ERK3 is expressed during thymocyte differentiation and that its expression is induced in mature peripheral T cells following activation of ERK1/2 by T-cell receptor (TCR) signalling. Herein, we have investigated whether ERK3 expression is required for proper T-cell selection. Using a knock-in mouse model in which the coding sequence of ERK3 is replaced by the gene encoding for the β-galactosidase reporter, we show that ERK3 is expressed by double-positive (DP) thymocytes undergoing positive selection. In ERK3-deficient mice with a polyclonal TCR repertoire, we observe a decrease in positive selection. This reduction in positive selection was also observed when ERK3-deficient mice were backcrossed to class I- and class II-restricted TCR transgenic mice. Furthermore, the response of DP thymocytes to in vitro TCR stimulation was strongly reduced in ERK3-deficient mice. Together, these results show that ERK3 expression following TCR signalling is critical for proper thymic positive selection.

Retroviral transduction of lineage antigen-negative (Lin−) cells: A valuable alternative for the generation of T cell receptor (TCR) retrogenic mice

Mice with virtually all T cells expressing a single T cell receptor (TCR) on their surface have been instrumental in understanding the development of immature thymocytes. For many years, such an engineering has been achieved essentially by inserting rearranged TCR α and β chain coding sequences into the genome through co-microinjection into fertilized eggs (TCR transgenesis). More recently, a novel methodology relying on the reconstitution of T cell deficient hosts with retrovirally-transduced multipotent bone marrow cells has been developed. Hence, TCR retrogenesis allows for the in vivo study of given TCR specificities in a faster and less expensive manner. While initial procedures were taking advantage of 5-Fluorouracil (5-FU) treatment of RAG-deficient or SCID donor mice as source of haematopoietic stem cells, we used bone marrow cell suspensions enriched in lineage antigen-negative (Lin−) cells from untreated donors for TCR retrogenesis. In contrast to cells from 5-FU-treated donors, transduced Lin− cells consistently generated a sizable retrogenic pool of thymocytes and required less donor mice. In such retrogenic mice, immature thymocytes bearing a major histocompatibility complex (MHC) class II-restricted TCR differentiated into the expected CD4 mature T cell lineage and populated the peripheral lymphoid organs where they retained the capacity to react to their cognate ligand. Lin− cell-enriched BM cells represent therefore, a reliable alternative to 5-FU treatment for retroviral transduction of haematopoietic stem cells and TCR retrogenic derivation.

Development of genetically engineered CD4+ and CD8+ T cells expressing TCRs specific for a M. tuberculosis 38-kDa antigen

Cell-mediated immunity is critical to the clearance of Mycobacterium tuberculosis due to the primarily intracellular niche of this pathogen. Adoptive transfer of M. tuberculosis-specific effector T cells has been shown to confer immunity to M. tuberculosis-infected recipients resulting in M. tuberculosis clearance. However, it is difficult to generate sufficient numbers of M. tuberculosis antigen-specific T cells in a short time. Recent studies have developed T cell receptor (TCR) gene-modified T cells that allow for the rapid generation of large numbers of antigen-specific T cells. Many TCRs that target various tumor and viral antigens have now been isolated and shown to have functional activity. Nevertheless, TCRs specific for intracellular bacterial antigens (including M. tuberculosis antigens) have yet to be isolated and their functionality confirmed. We isolated M. tuberculosis 38-kDa antigen-specific HLA class I and class II-restricted TCRs and modified the TCR gene C regions by substituting nine amino acids with their murine TCR homologs (minimal murinization). Results showed that both wild-type and minimal murinized TCR genes were successfully cloned into retroviral vectors and transduced into primary CD4(+) and CD8(+) T cells and displayed anti-M. tuberculosis activity. As expected, minimal murinized TCRs displayed higher cell surface expression levels and stronger anti-M. tuberculosis activity than wild-type TCRs. To the best of our knowledge, this is the first report describing TCRs targeting M. tuberculosis antigens and this investigation provides the basis for future TCR gene-based immunotherapies that can be designed for the treatment of immunocompromised M. tuberculosis-infected patients.

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.

Crystal structure of an autoimmune TCR with optimal binding to its self-antigen - an alternative strategy for escaping negative selection (93.32)

Abstract The failure to eliminate self-reactive T cells during thymic selection is a prerequisite for autoimmunity. To escape deletion, autoreactive T cell receptors (TCRs) usually form relatively unstable complexes with their self-peptide/MHC ligands. As shown previously, these TCRs adopt suboptimal binding topologies as opposed to anti-microbial TCRs. In other cases, escape can be mediated by weak binding between self-peptides and MHC. Here, we determined the crystal structure, to 2.7 Å resolution, of a human MHC class II-restricted autoimmune TCR (3C8) bound to its self-antigen, MBP 114-126/HLA-DR4. TCR 3C8, which was originally isolated from a multiple sclerosis patient, has demonstrated pathogenic potential in humanized transgenic mice. The MBP self-peptide binds HLA-DR4 with unusually low affinity. The structure showed a loose accommodation of MBP in the peptide binding groove. By contrast, 3C8 binds to MBP/HLA-DR4 at the high end of the affinity range for TCR-peptide/MHC interactions. Moreover, 3C8 engages its self-ligand via a docking mode that closely resembles the optimal binding topology of anti-foreign TCRs, but is distinct from that of other autoreactive TCRs. Thus, the 3C8-MBP/HLA-DR4 complex reveals the structural basis for an alternative strategy by which autoreactive T cells escape negative selection, yet still retain the ability to become sufficiently activated in the periphery to be pathogenic.

A New Twist in TCR Diversity Revealed by a Forbidden αβ TCR

We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A u-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the Vα/Vβ interface exert indirect effects on recognition by influencing the Vα/Vβ interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the Vα/Vβ interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.

Seeing the change: TCR binds pMHC with an ‘induced-fit’

At the molecular level, T-cell recognition of the peptide–MHC (pMHC) on antigen-presenting cells (APCs) is orchestrated by several protein–protein interactions on the surfaces of both cells. Among them, the interaction of T-cell receptor (TCR) on the surface of T cells with its ligand, pMHC, is characterized by low-affinity, slow kinetics and a high degree of cross-reactivity. Based on only one available bound and unbound complex crystal structure of TCR–pMHC (2C TCR with H-2Kb), with some thermodynamic analysis (both class I- and class II-restricted TCRs), and one case of NMR structural analysis (D10 TCR), ‘induced fit’ or ‘local conformational readjustment’ has been proposed for the interaction between TCR and pMHC. Reiser et al. [ 1. Reiser J.B. et al. A T-cell receptor CDR3β loop undergoes conformational changes of unprecedented magnitude upon binding to a peptide–MHC class I complex. Immunity. 2002; 16: 345-354 Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar ] now provide new evidence that local conformational readjustment does occur in the TCR–pMHC interface, upon binding of the TCR with pMHC. The authors even suggest calling this readjustment ‘conformational change’ to fit the binding.

Antagonist peptide selects thymocytes expressing a class II major histocompatibility complex-restricted T cell receptor into the CD8 lineage.

CD4/CD8 lineage decision is an important event during T cell maturation in the thymus. CD8 T cell differentiation usually requires corecognition of major histocompatibility complex (MHC) class I by the T cell receptor (TCR) and CD8, whereas CD4 T cells differentiate as a consequence of MHC class II recognition by the TCR and CD4. The involvement of specific peptides in the selection of T cells expressing a particular TCR could be demonstrated so far for the CD8 lineage only. We used mice transgenic for an MHC class II-restricted TCR to investigate the role of antagonistic peptides in CD4 T cell differentiation. Interestingly, antagonists blocked the development of CD4(+) cells that normally differentiate in thymus organ culture from those mice, and they induced the generation of CD8(+) cells in thymus organ culture from mice impaired in CD4(+) cell development (invariant chain-deficient mice). These results are in line with recent observations that antagonistic signals direct differentiation into the CD8 lineage, regardless of MHC specificity.

Affinity and kinetics of the interactions between an αβ T-cell receptor and its superantigen and class II-MHC/peptide ligands

Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR β-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted αβ TCR and its superantigen and MHC/peptide ligands. The extracellular domains of the murine D10 TCR (Vα2, Vβ8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble D10 TCR bound to Staphylococcus aureus enterotoxin C2 with an association rate of 1.69 ± 0.12 × 10 4M −1 sec −1 and a dissociation rate of 1.9 ± 0.47 × 10 −2sec −1, giving a dissociation constant of 1.1 μM. Binding of the TCR to S. aureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-A k molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I-A k chimera specifically stimulated proliferation of the D 10 T-cell clone, and bound to immobilized soluble D10 TCR with an association rate of 1.07 ± 0.19 × 10 4M −1sec −1 and a dissociation rate of 2.2 ± 0.65 × 10 −2sec −1, giving a dissociation constant of 2.1 μM.