RESUMEN
Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer.
Asunto(s)
Complejo CD3/metabolismo , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Secuencias de Aminoácidos/inmunología , Animales , Complejo CD3/inmunología , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Caveolin-1 (Cav1) regulates the nanoscale organization and compartmentalization of the plasma membrane. Here we found that Cav1 controlled the distribution of nanoclusters of isotype-specific B cell antigen receptors (BCRs) on the surface of B cells. In mature B cells stimulated with antigen, the immunoglobulin M BCR (IgM-BCR) gained access to lipid domains enriched for GM1 glycolipids, by a process that was dependent on the phosphorylation of Cav1 by the Src family of kinases. Antigen-induced reorganization of nanoclusters of IgM-BCRs and IgD-BCRs regulated BCR signaling in vivo. In immature Cav1-deficient B cells, altered nanoscale organization of IgM-BCRs resulted in a failure of receptor editing and a skewed repertoire of B cells expressing immunoglobulin-µ heavy chains with hallmarks of poly- and auto-reactivity, which ultimately led to autoimmunity in mice. Thus, Cav1 emerges as a cell-intrinsic regulator that prevents B cell-induced autoimmunity by means of its role in plasma-membrane organization.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Caveolina 1/metabolismo , Tolerancia Inmunológica , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Caveolina 1/genética , Expresión Génica , Tolerancia Inmunológica/genética , Inmunoglobulina D/inmunología , Inmunoglobulina D/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Unión Proteica , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.
Asunto(s)
Inmunidad Adaptativa , Colesterol/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/inmunología , Regulación Alostérica , Antígenos/inmunología , Antígenos/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Humanos , Células Jurkat , Activación de Linfocitos , Modelos Inmunológicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Transducción de SeñalRESUMEN
Caveolin-1 (Cav-1) is a key organizer of membrane specializations and a scaffold protein that regulates signaling in multiple cell types. We found increased Cav-1 expression in human and murine T cells after allogeneic hematopoietic cell transplantation. Indeed, Cav-1(-/-)donor T cells caused less severe acute graft-versus-host disease (GVHD) and yielded higher numbers of regulatory T cells (Tregs) compared with controls. Depletion of Tregs from the graft abrogated this protective effect. Correspondingly, Treg frequencies increased when Cav-1(-/-)T cells were exposed to transforming growth factor-ß/T-cell receptor (TCR)/CD28 activation or alloantigen stimulation in vitro compared with wild-type T cells. Mechanistically, we found that the phosphorylation of Cav-1 is dispensable for the control of T-cell fate by using a nonphosphorylatable Cav-1 (Y14F/Y14F) point-mutation variant. Moreover, the close proximity of lymphocyte-specific protein tyrosine kinase (Lck) to the TCR induced by TCR-activation was reduced in Cav-1(-/-)T cells. Therefore, less TCR/Lck clustering results in suboptimal activation of the downstream signaling events, which correlates with the preferential development into a Treg phenotype. Overall, we report a novel role for Cav-1 in TCR/Lck spatial distribution upon TCR triggering, which controls T-cell fate toward a regulatory phenotype. This alteration translated into a significant increase in the frequency of Tregs and reduced GVHD in vivo.
Asunto(s)
Caveolina 1/metabolismo , Caveolina 1/fisiología , Regulación de la Expresión Génica , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/citología , Caveolina 1/genética , Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , Estudios Prospectivos , Transducción de Señal , Linfocitos T Reguladores/citología , Factor de Crecimiento Transformador beta/metabolismo , Trasplante HomólogoRESUMEN
Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos/inmunología , Proteínas Oncogénicas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Sitios de Unión , Complejo CD3/metabolismo , Línea Celular Tumoral , Humanos , Células Jurkat , Proteínas Oncogénicas/genética , Fosforilación , Dominios Proteicos Ricos en Prolina , Unión Proteica , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Dominios Homologos srcRESUMEN
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.
Asunto(s)
Complejo CD3 , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Transducción de Señal , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejo CD3/metabolismo , Células HEK293 , Motivo de Activación del Inmunorreceptor Basado en Tirosina , Células Jurkat , Activación de Linfocitos/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Fosforilación , Unión Proteica , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Transducción de Señal/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Proteína Tirosina Quinasa ZAP-70/genéticaRESUMEN
Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.
Asunto(s)
Síndromes de Inmunodeficiencia , Linfopenia , Lactante , Humanos , Animales , Ratones , Antígenos CD28 , Linfocitos T CD4-Positivos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Receptores de Antígenos de Linfocitos T/genética , Inflamación/genética , Linfopenia/genéticaRESUMEN
The T cell antigen receptor (TCR) is expressed on T cells, which orchestrate adaptive immune responses. It is composed of the ligand-binding clonotypic TCRαß heterodimer and the non-covalently bound invariant signal-transducing CD3 complex. Among the CD3 subunits, the CD3ε cytoplasmic tail contains binding motifs for the Src family kinase, Lck, and the adaptor protein, Nck. Lck binds to a receptor kinase (RK) motif and Nck binds to a proline-rich sequence (PRS). Both motifs only become accessible upon ligand binding to the TCR and facilitate the recruitment of Lck and Nck independently of phosphorylation of the TCR. Mutations in each of these motifs cause defects in TCR signaling and T cell activation. Here, we investigated the role of Nck in proximal TCR signaling by silencing both Nck isoforms, Nck1 and Nck2. In the absence of Nck, TCR phosphorylation, ZAP70 recruitment, and ZAP70 phosphorylation was impaired. Mechanistically, this is explained by loss of Lck recruitment to the stimulated TCR in cells lacking Nck. Hence, our data uncover a previously unknown cooperative interaction between Lck and Nck to promote optimal TCR signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Oncogénicas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Complejo CD3/metabolismo , Humanos , Células Jurkat , Fosforilación , Unión Proteica , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
T lymphocytes expressing the γδ T cell receptor (γδ TCR) can recognize antigens expressed by tumor cells and subsequently kill these cells. γδ T cells are indeed used in cancer immunotherapy clinical trials. The anti-CD3ε antibody UCHT1 enhanced the in vitro tumor killing activity of human γδ T cells by an unknown molecular mechanism. Here, we demonstrate that Fab fragments of UCHT1, which only bind monovalently to the γδ TCR, also enhanced tumor killing by expanded human Vγ9Vδ2 γδ T cells or pan-γδ T cells of the peripheral blood. The Fab fragments induced Nck recruitment to the γδ TCR, suggesting that they stabilized the γδ TCR in an active CD3ε conformation. However, blocking the Nck-CD3ε interaction in γδ T cells using the small molecule inhibitor AX-024 neither reduced the γδ T cells' natural nor the Fab-enhanced tumor killing activity. Likewise, Nck recruitment to CD3ε was not required for intracellular signaling, CD69 and CD25 up-regulation, or cytokine secretion by γδ T cells. Thus, the Nck-CD3ε interaction seems to be dispensable in γδ T cells.
RESUMEN
Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F-myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.
Asunto(s)
Antígeno B7-H1/metabolismo , Neoplasias Hematológicas/metabolismo , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/metabolismo , Animales , Antígeno B7-H1/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Transformación Celular Neoplásica , Neoplasias Hematológicas/genética , Humanos , Janus Quinasa 2/genética , Células K562 , Ratones , Trastornos Mieloproliferativos/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Activation of the T cell receptor (TCR) by antigen is the key step in adaptive immunity. In the αßTCR, antigen induces a conformational change at the CD3 subunits (CD3 CC) that is absolutely required for αßTCR activation. Here, we demonstrate that the CD3 CC is not induced by antigen stimulation of the mouse G8 or the human Vγ9Vδ2 γδTCR. We find that there is a fundamental difference between the activation mechanisms of the αßTCR and γδTCR that map to the constant regions of the TCRαß/γδ heterodimers. Enforced induction of CD3 CC with a less commonly used monoclonal anti-CD3 promoted proximal γδTCR signaling but inhibited cytokine secretion. Utilizing this knowledge, we could dramatically improve in vitro tumor cell lysis by activated human γδ T cells. Thus, manipulation of the CD3 CC might be exploited to improve clinical γδ T cell-based immunotherapies.