Phospho-mimetic CD3ε variants prevent TCR and CAR signaling.

Introduction: Antigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome. Methods: We mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM. Results: Integrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK. Conclusions: The data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CAR signaling and highlights the key role of CD3ε signalosome to tune signal transduction.

Enhancing the Antitumor Immunity of T Cells by Engineering the Lipid-Regulatory Site of the TCR/CD3 Complex.

The engagement of the T-cell receptor (TCR) by a specific peptide-MHC ligand initiates transmembrane signaling to induce T-cell activation, a key step in most adaptive immune responses. Previous studies have indicated that TCR signaling is tightly regulated by cholesterol and its sulfate metabolite, cholesterol sulfate (CS), on the membrane. Here, we report a novel mechanism by which CS modulates TCR signaling through a conformational change of CD3 subunits. We found that the negatively charged CS interacted with the positively charged cytoplasmic domain of CD3ε (CD3εCD) to enhance its binding to the cell membrane and induce a stable secondary structure. This secondary structure suppressed the release of CD3εCD from the membrane in the presence of Ca2+, which in turn inhibited TCR phosphorylation and signaling. When a point mutation (I/A) was introduced to the intracellular immunoreceptor tyrosine-based activation motifs (YxxI-x6-8-YxxL) of CD3ε subunit, it reduced the stability of the secondary structure and regained sensitivity to Ca2+, which abolished CS-mediated inhibition and enhanced the signaling of the TCR complex. Notably, the I/A mutation could be applied to both murine and human TCR-T cell therapy to improve the antitumor efficacy. Our study reveals insights into the regulatory mechanism of TCR signaling and provides a strategy to functionally engineer the TCR/CD3 complex for T cell-based cancer immunotherapy.

CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.

Lck bound to coreceptor is less active than free Lck.

Src family kinase Lck plays critical roles during T cell development and activation, as it phosphorylates the TCR/CD3 complex to initiate TCR signaling. Lck is present either in coreceptor-bound or coreceptor-unbound (free) forms, and we here present evidence that the two pools of Lck have different molecular properties. We discovered that the free Lck fraction exhibited higher mobility than CD8α-bound Lck in OT-I T hybridoma cells. The free Lck pool showed more activating Y394 phosphorylation than the coreceptor-bound Lck pool. Consistent with this, free Lck also had higher kinase activity, and free Lck mediated higher T cell activation as compared to coreceptor-bound Lck. Furthermore, the coreceptor-Lck coupling was independent of TCR activation. These findings give insights into the initiation of TCR signaling, suggesting that changes in coreceptor-Lck coupling constitute a mechanism for regulation of T cell sensitivity.

A scoring system for the electrostatic complementarities of T-cell receptors and cancer-mutant amino acids: multi-cancer analyses of associated survival rates.

The anti-tumor immune response is considered to be due to the T-cell receptor (TCR) binding to tumor antigens, which can be either wild-type, early stem cell proteins, presumably foreign to a developed immune system; or mutant peptides, foreign to the immune system because of a mutant amino acid (aa) or otherwise somatically altered aa sequence. Recently, very large numbers of TCR complementarity-determining region-3 (CDR3) aa sequences obtained from tumor specimens have become available. We developed a novel algorithm for assessing the complementarity of tumor mutant peptides and TCR CDR3s, based on the retrieval of TCR CDR3 aa sequences from both tumor specimen and patient blood exomes and by using an automated process of assessing CDR3 and mutant aa electrical charges. Results indicated many instances where high electrostatic complementarity was associated with a higher survival rate. In particular, our approach led to the identification of specific genes contributing significantly to the complementary, TCR CDR3-mutant aa. These results suggest a novel approach to tumor immunoscoring and may lead to the identification of high-priority neo-antigen, peptide vaccines; or to the identification of ex vivo stimulants of tumor-infiltrating lymphocytes.

Actin polymerization regulates recruitment of Nck to CD3ε upon T-cell receptor triggering.

Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε.

Compositional characteristics of human peripheral TRBV pseudogene rearrangements.

The diversity of the T cell receptor (TCR) complementarity determining region 3 (CDR3) repertoire is the result of random combinations, insertions and deletions during recombination of the germline V, D and J gene fragments. During evolution, some human TCR beta chain variable (TRBV) pseudogenes have been retained. Many previous studies have focused on functional TRBV genes, while little attention has been given to TRBV pseudogenes. To describe the compositional characteristics of TRBV pseudogene rearrangements, we compared and analysed TRBV pseudogenes, TRBV open reading frames (ORFs) and functional TRBV genes via high-throughput sequencing of DNA obtained from the peripheral blood of 4 healthy volunteers and 4 patients. Our results revealed several differences in J and D gene usage. The V deletion distribution profile of the pseudogenes was significantly different from that of the ORFs and functional genes. In addition, arginine, lysine and cysteine were more frequently used in putative CDR3 pseudogene rearrangements, while functional rearrangements used more leucine. This study presents a comprehensive description of the compositional characteristics of peripheral TRBV pseudogene rearrangements, which will provide a reference for further research on TRBV pseudogenes.

Phase 1 Results of ZUMA-1: A Multicenter Study of KTE-C19 Anti-CD19 CAR T Cell Therapy in Refractory Aggressive Lymphoma.

Outcomes for patients with refractory diffuse large B cell lymphoma (DLBCL) are poor. In the multicenter ZUMA-1 phase 1 study, we evaluated KTE-C19, an autologous CD3ζ/CD28-based chimeric antigen receptor (CAR) T cell therapy, in patients with refractory DLBCL. Patients received low-dose conditioning chemotherapy with concurrent cyclophosphamide (500 mg/m2) and fludarabine (30 mg/m2) for 3 days followed by KTE-C19 at a target dose of 2 × 106 CAR T cells/kg. The incidence of dose-limiting toxicity (DLT) was the primary endpoint. Seven patients were treated with KTE-C19 and one patient experienced a DLT of grade 4 cytokine release syndrome (CRS) and neurotoxicity. Grade ≥3 CRS and neurotoxicity were observed in 14% (n = 1/7) and 57% (n = 4/7) of patients, respectively. All other KTE-C19-related grade ≥3 events resolved within 1 month. The overall response rate was 71% (n = 5/7) and complete response (CR) rate was 57% (n = 4/7). Three patients have ongoing CR (all at 12+ months). CAR T cells demonstrated peak expansion within 2 weeks and continued to be detectable at 12+ months in patients with ongoing CR. This regimen of KTE-C19 was safe for further study in phase 2 and induced durable remissions in patients with refractory DLBCL.

Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes.

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that ß2 microglobulin (ß2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15-23 (InsB15-23) to the N terminus of ß2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/ß2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/ß2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15-23/ß2m/CD3-ζ mRNA was activated by an InsB15-23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15-23/ß2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206-214 (IGRP206-214)/ß2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15-23/ß2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes.

A conserved αß transmembrane interface forms the core of a compact T-cell receptor-CD3 structure within the membrane.

The T-cell antigen receptor (TCR) is an assembly of eight type I single-pass membrane proteins that occupies a central position in adaptive immunity. Many TCR-triggering models invoke an alteration in receptor complex structure as the initiating event, but both the precise subunit organization and the pathway by which ligand-induced alterations are transferred to the cytoplasmic signaling domains are unknown. Here, we show that the receptor complex transmembrane (TM) domains form an intimately associated eight-helix bundle organized by a specific interhelical TCR TM interface. The salient features of this core structure are absolutely conserved between αß and γδ TCR sequences and throughout vertebrate evolution, and mutations at key interface residues caused defects in the formation of stable TCRαß:CD3δε:CD3γε:ζζ complexes. These findings demonstrate that the eight TCR-CD3 subunits form a compact and precisely organized structure within the membrane and provide a structural basis for further investigation of conformationally regulated models of transbilayer TCR signaling.

Dendritic cells promote expansion and survival of aberrant TCR-negative intraepithelial lymphocyte lines from refractory celiac disease type II patients.

Celiac disease (CD) patients who fail to respond to a gluten-free diet suffer from refractory celiac disease (RCD). A marked expansion of intraepithelial lymphocytes (IEL) lacking surface TCR/CD3 expression characterizes the RCD subtype II. In up to 50% of RCDII patients these so-called aberrant IEL (a-IEL) develop into lymphoma and can disseminate into other tissues. Elevated levels of Interleukin-15 (IL-15) in the intestine of CD and RCD patients likely contribute to the expansion of a-IEL. Here, we investigated if interactions with other cells might also influence a-IEL expansion. Similar to IL-15, cells from the monocyte lineage, particularly mature dendritic cells (DCs), promoted proliferation, prevented apoptosis and induced IFNγ secretion of a-IEL derived from RCDII biopsies (RCDII cell lines), which in turn induced CXCL10. In contrast to IL-15, mature DCs did not induce proliferation of regular TCR(+)IEL lines, generated from CD biopsies and IL-15-blocking antibodies did not inhibit DC-induced proliferation of RCDII cell lines. Furthermore, proliferation was dependent on cell-cell contact, but independent of the HLA-genotype of the stimulating cells. Our results suggest that contact with DC, either in the epithelium or upon dissemination, contributes to uncontrolled expansion of a-IEL in RCDII, independent of HLA-genotype and IL-15.

Guanylate binding protein 1-mediated interaction of T cell antigen receptor signaling with the cytoskeleton.

GTPases act as important switches in many signaling events in cells. Although small and heterotrimeric G proteins are subjects of intensive studies, little is known about the large IFN-inducible GTPases. In this article, we show that the IFN-γ-inducible guanylate binding protein 1 (GBP-1) is a regulator of T cell activation. Silencing of GBP-1 leads to enhanced activation of early T cell Ag receptor/CD3 signaling molecules, including Lck, that is translated to higher IL-2 production. Mass spectrometry analyses showed that regulatory cytoskeletal proteins, like plastin-2 that bundles actin fibers and spectrin ß-chain, brain 1 that links the plasma membrane to the actin cytoskeleton, are binding partners of GBP-1. The spectrin cytoskeleton influences cell spreading and surface expression of TCR/CD3 and the leukocyte phosphatase CD45. We found higher cell spreading and enhanced surface expression of TCR/CD3 and CD45 in GBP-1 silenced T cells that explain their enhanced TCR/CD3 signaling. We conclude that GBP-1 is a downstream processor of IFN-γ via which T cells regulate cytoskeleton-dependent cell functions.

T cells redirected by a CD3ζ chimeric antigen receptor can establish self-antigen-specific tumour protection in the long term.

A majority of cancer deaths are because of an uncontrolled relapse of the disease despite initial remission after therapy, asking for strategies to control tumour cells in the long term. Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed promising success in primary tumour elimination; the capacity of such engineered T cells to establish enduring tumour protection is currently a matter of discussion, in particular as most targeted 'tumour-associated antigens' are self-antigens. To address the issue in a clinically relevant model that closely mimics the human situation, we recorded rejection of carcinoembryonic antigen (CEA)-positive pancreatic tumours in the CEA transgenic mouse that expressed CEA as self-antigen in healthy cells of the gastrointestinal tract. Adoptive therapy with CD8(+) T cells, which were redirected by a CEA-specific, low-affinity CAR with CD3ζ endodomain, eliminated CEA(+) tumours in a primary response; cured mice produced an efficient recall response in the long term towards CEA(+) tumour cells upon rechallenge. Secondary tumour rejection was CEA specific, mediated by engineered T cells and did not require host T cells. No toxicity towards healthy tissues with CEA expression was recorded. Data indicate that adoptive therapy with engineered T cells can establish self-antigen-specific tumour protection in the long term without autoimmunity.

A change in CD3γ, CD3δ, CD3ϵ, and CD3ζ gene expression in T-lymphocytes from benzene-exposed and benzene-poisoned workers.

Benzene is known to be highly toxic to a variety of cell types, including lymphocytes. A previous study showed that T-lymphocyte immune function disorder might be related to benzene exposure. To elucidate characteristics of TCR signal transduction in benzene-exposed workers, expression levels of CD3γ, CD3δ, CD3ϵ, and CD3ζ genes in peripheral blood mononuclear cells (PBMC) were analyzed. Real-time RT-PCR using SYBR Green I was used to detect CD3 gene expression levels in PBMC from 20 benzene-exposed workers, seven workers with chronic mild benzene poisoning, five workers with chronic severe benzene poisoning and 14 healthy individuals (controls). The relative mRNA expression level was analyzed by the 2(-Δct) × 100% method. In benzene-exposed worker cells, significantly higher CD3δ, CD3ϵ, and CD3ζ, expression levels were observed as compared with values for cells from the healthy controls. In the workers with chronic severe benzene poisoning, lymphocyte CD3γ, CD3ϵ, and CD3ζ gene expression levels were significantly lower than in control cells. Lymphocytes from chronic mildly benzene- poisoned workers evinced two different gene expression patterns, i.e., CD3γ and CD3ϵ levels were similar to those in the benzene-exposed worker cells, but CD3δ and CD3ζ expression levels were significantly lower relative to those in cells from chronic severely-benzene-poisoned counterparts. It remained to be determined if these reductions in expression of these genes presage or are indicative of deficiencies in the activities of T-lymphocytes in these workers. For now, it is hoped that this study may contribute to a better understanding of the disorders in cellular immunity frequently found with benzene-exposed workers.

Change in expression pattern of TCR-CD3 complex in patients with multiple myeloma.

In haematological malignancy, cell-mediated immunity has been shown to be suppressed in advanced disease. This immune dysfunction may be due, in part, to altered expression of the T cell receptor (TCR)-CD3 complex. The distribution and clonality of the TCR Vbeta repertoire and the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in T cells from patients with multiple myeloma (MM) were investigated. Specific Vbeta subfamily primers, reverse transcription polymerase chain reaction, and the GeneScan® technique were used to analyse the expression of the TCR Vbeta subfamily and the clonality of Vbeta T cells in 11 patients with MM. Real-time reverse transcription polymerase chain reaction was used to detect the expression levels of CD3gamma, CD3delta, CD3epsilon, and CD3zeta genes in peripheral blood mononuclear cells from 19 patients with MM. The beta2-microglobulin gene was used as an endogenous reference. A total of 5-22 Vbeta subfamily T cells were detected in different patients (mean value of expressed Vbeta subfamilies was 12.55±6.11), whereas all 24 Vbeta genes were identified in all control samples. The most frequently expressed Vbeta subfamilies were Vbeta1 (100%), Vbeta2, Vbeta3, Vbeta9, Vbeta13, and Vbeta16 (81.8%), while the expression of Vbeta20 was undetectable in all MM samples. Oligoclonal expansion of one or more Vbeta subfamily of T cells was detected in all patients. Such expansions involved different MM stages, and the numbers of expanded clonal Vbeta subfamilies seemed higher in stage I/II groups than in stage III; however, there was no significant difference. Among MM samples, of the Vbeta subfamily members, Vbeta13, Vbeta1, and Vbeta21, were expanded most frequently. A significant decrease in the expression level of the CD3gamma gene was observed in MM samples; in contrast, a higher expression of CD3epsilon was found in the MM group than in the healthy group. The expression pattern of the four CD3 chains was epsilon>zeta>delta>gamma in peripheral blood mononuclear cells from MM, while a gamma>epsilon>zeta>delta expression pattern was found in healthy controls. In conclusions, the present study presents precise data on changes in the variability of Vbeta patterns and expression of TCR signal transduction molecules in MM patients compared to controls, which may be associated with immune dysfunction. This study contributes to a better understanding of the cellular immune features in MM patients.

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and ß chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.

Inhibition of T cell activation through down-regulation of TCR-CD3 expression mediated by an anti-CD90 Ab.

We are trying to develop new Abs that can manipulate CD4 T cell responses and are usable as immunosuppressive agents. To this end, we performed functional screening, in which we examined the effect of an Ab on the proliferation of mouse CD4 T cells upon activation. The Ab, LP5, inhibited the activation of CD4 T cells stimulated with an anti-CD3 Ab or peptide antigen. The Ab alone had no stimulatory effect on CD4 T cells. Biochemical experiments demonstrated that LP5 recognized the Thy-1 (CD90) molecule. Interestingly, the treatment of CD4 T cells with LP5 in vitro induced a temporary down-regulation of CD3 expression at the cell surface. TCR molecules were also affected. Other anti-CD90 Abs not inhibitory to CD4 T cell activation failed to induce a reduction in CD3. Experiments in vitro revealed that the down-regulation caused by LP5 is due to an accelerated endocytosis of cell surface CD3. In addition, it was shown that CD3 down-regulation before or in the early stages of T cell activation is critical for the induction of hyporesponsiveness. Experiments in vivo showed that pre-treatment of CD4 T cells with LP5 inhibited the rejection of semi-allogeneic bone marrow transplants. Based on these observations, we propose that CD3 down-regulation without any stimulatory activity against T cells could be one approach to inhibiting T cell activation, and CD90 would be an appropriate target.

Basal and antigen-induced exposure of the proline-rich sequence in CD3ε.

The CD3ε cytoplasmic tail contains a conserved proline-rich sequence (PRS) that influences TCR-CD3 expression and signaling. Although the PRS can bind the SH3.1 domain of the cytosolic adapter Nck, whether the PRS is constitutively available for Nck binding or instead represents a cryptic motif that is exposed via conformational change upon TCR-CD3 engagement (CD3Δc) is currently unresolved. Furthermore, the extent to which a cis-acting CD3ε basic amino acid-rich stretch (BRS), with its unique phosphoinositide-binding capability, might impact PRS accessibility is not clear. In this study, we found that freshly harvested primary thymocytes expressed low to moderate basal levels of Nck-accessible PRS ("open-CD3"), although most TCR-CD3 complexes were inaccessible to Nck ("closed-CD3"). Ag presentation in vivo induced open-CD3, accounting for half of the basal level found in thymocytes from MHC(+) mice. Additional stimulation with either anti-CD3 Abs or peptide-MHC ligands further elevated open-CD3 above basal levels, consistent with a model wherein antigenic engagement induces maximum PRS exposure. We also found that the open-CD3 conformation induced by APCs outlasted the time of ligand occupancy, marking receptors that had been engaged. Finally, CD3ε BRS-phosphoinositide interactions played no role in either adoption of the initial closed-CD3 conformation or induction of open-CD3 by Ab stimulation. Thus, a basal level of open-CD3 is succeeded by a higher, induced level upon TCR-CD3 engagement, involving CD3Δc and prolonged accessibility of the CD3ε PRS to Nck.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

[A study of T-cell reconstitution after HLA-matched sibling bone marrow transplantation in leukemia patients].

OBJECTIVE: To study T-cell reconstitution by the assessment of T-cell receptor excision circle (TRECs) and T-cell receptor clonal repertoire after HLA-matched sibling bone marrow transplantation (MSD-BMT) in leukemia patients and try to reveal the rule of T-cells repopulation in MSD-BMT. METHODS: Real-time quantitative PCR detection of TRECs was carried out in the DNA of peripheral blood mononuclear cells from 23 leukemia patients who underwent MSD-BMT. The content of TRECs in 70 normal donor individuals was also detected to determine the normal range of TRECs in healthy subjects. Among them, 10 patients received RT-PCR to amplify 24 subfamily genes of T-cell receptor B variable (TCRBV) and five normal donors served as control. The PCR products were further analyzed with genescan to evaluate the clonality of BV subfamily and calculate the usage rate of BV families. RESULTS: The mean value of TRECs in normal donors was (3351.1 +/- 3711.1) copies/10(5) cells. There was an inverse correlation between the value of TRECs and the age of healthy subjects. Before transplantation, all the patients were detected for the number of TRECs, the mean TRECs number was (307.9 +/- 433.3) copies/10(5) cells and it was far lower than that of healthy subjects. From one month to five months after bone marrow transplantative (BMT), the TRECs levels were low and in some patients they even could not be detected. Six months later, TRECs levels increased obviously and maintained that high nearly one year. 24 months after BMT, the number of TRECs increased and reached the pretransplantation status. One patient was detected 2 m and 3 m after transplantation and found that there was a tendency of additional use of BV families and an increase of the expression of CDR3 polymorphism. 2-15 months after transplantation, in ten of the patients, there were 7-16 BV subfamilies expressing and in more than 48% the expression was polyclinic. Monoclones and oligoclones existed in 24 BV subfamilies. CONCLUSIONS: At an early stage after BMT, the number of TRECs was low and remained so for a long time. TCRBV CDR3 repertoire showed certain BV families expressing. 6 months after BMT, thymus produced certain number of naive T cells and TCRBV CDR3 repertoire showed additional use of BV families and increased expression of polymorphism.