RESUMEN
Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.
Asunto(s)
Inmunoterapia Adoptiva , Linfocitos T , Inmunoterapia Adoptiva/métodos , Inmunoterapia , Receptores de Antígenos de Linfocitos TRESUMEN
CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs.
Asunto(s)
Receptores Quiméricos de Antígenos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Inmunoterapia Adoptiva/métodos , Linfocitos T/metabolismoRESUMEN
Large-scale target cell isolation from patient blood preparations is one of the critical operations during drug product manufacturing for personalized cell therapy in immuno-oncology. Use of high-affinity murine antibody coated magnetic nanoparticles that remain on isolated cells is the current standard applied for this purpose. Here, we present the transformation of previously described technology - non-magnetic immunoaffinity column chromatography-based cell selection with reversible reagents into a new clinical-grade cell isolation platform called Automated Traceless Cell affinity chromatography (ATC). ATC is a fully closed and GMP-compliant cell selection and manufacturing system. Reversibility of reagents enables (sequential) positive cell selection, optionally in combination with depletion columns, enabling capture of highly specific cell subsets. Moreover, synergy with other Streptamer-based technologies allows novel uses beyond cell isolation including integrated and automated on-column target cell activation. In conclusion, ATC technology is an innovative as well as versatile platform to select, stimulate and modify cells for clinical manufacturing and downstream therapies.
Asunto(s)
Cromatografía , Animales , Separación Celular/métodos , Humanos , RatonesRESUMEN
T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.
Asunto(s)
Indicadores y Reactivos/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Animales , Proliferación Celular , Perfilación de la Expresión Génica , Humanos , Inmunoterapia Adoptiva , Ratones , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Therapeutic T cells with desired specificity can be engineered by introducing T-cell receptors (TCRs) specific for antigens of interest, such as those from pathogens or tumour cells. However, TCR engineering is challenging, owing to the complex heterodimeric structure of the receptor and to competition and mispairing between endogenous and transgenic receptors. Additionally, conventional TCR insertion disrupts the regulation of TCR dynamics, with consequences for T-cell function. Here, we report the outcomes and validation, using five different TCRs, of the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) with non-virally delivered template DNA for the elimination of endogenous TCR chains and for the orthotopic placement of TCRs in human T cells. We show that, whereas the editing of a single receptor chain results in chain mispairing, simultaneous editing of α- and ß-chains combined with orthotopic TCR placement leads to accurate αß-pairing and results in TCR regulation similar to that of physiological T cells.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Antígenos de Neoplasias/inmunología , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Técnicas de Inactivación de Genes , Genes Codificadores de los Receptores de Linfocitos T/genética , Vectores Genéticos/genética , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Retroviridae/genética , Transducción Genética , TransgenesRESUMEN
ζ-associated protein of 70 kDa (Zap70) is crucial for T-cell receptor (TCR) signaling. Loss of Zap70 in both humans and mice results in severe immunodeficiency. On the other hand, the expression of Zap70 in B-cell malignancies correlates with the severity of the disease. Because of its role in immune-related disorders, Zap70 has become a therapeutic target for the treatment of human diseases. It is well-established that the activity/expression of Zap70 is regulated by post-translational modifications of crucial amino acids including the phosphorylation of tyrosines and the ubiquitination of lysines. Here, we have investigated whether also oxidation of cysteine residues regulates Zap70 functions. We have identified C575 as a major sulfenylation site of Zap70. A C575A substitution results in protein instability, reduced activity, and increased dependency on the Hsp90/Cdc37 chaperone system. Indeed, Cdc37 overexpression reconstituted partially the expression but fully the function of Zap70C575A. C575 lies within a Mx(2)CWx(6)R motif which is highly conserved among almost all human tyrosine kinases. Mutation of any of the conserved amino acids, but not of a non-conserved residue preceding the cysteine, also results in Zap70 instability. Collectively, we have identified a new redox-active motif which is crucial for the regulation of Zap70 stability/activity. We believe that this motif has the potential to become a novel target for the development of therapeutic tools to modulate the expression/activity of kinases.
Asunto(s)
Secuencias de Aminoácidos/genética , Secuencia Conservada , Dominios y Motivos de Interacción de Proteínas/genética , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Mutación , Oxidación-Reducción , Unión Proteica , Estabilidad Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Proteína Tirosina Quinasa ZAP-70/químicaRESUMEN
The enzymatic activity of the Src family tyrosine kinase p56Lck (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr394 and Tyr505 Phosphorylation of Tyr394 and the conformational opening of Lck are believed to activate the kinase, whereas Tyr505 phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr394 to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr394 occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr394 plays a role in Lck function that goes beyond inducing an open conformation of the kinase.
Asunto(s)
Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Células Jurkat , Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Microscopía Confocal , Microscopía Fluorescente , Mutación , Fosforilación , Conformación Proteica , Linfocitos T/metabolismo , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
Dendritic cells (DCs) are a heterogeneous group of mononuclear phagocytes with versatile roles in immunity. They are classified predominantly based on phenotypic and functional properties, namely their stellate morphology, expression of the integrin CD11c, and major histocompatibility class II molecules, as well as their superior capacity to migrate to secondary lymphoid organs and stimulate naïve T cells. However, these attributes are not exclusive to DCs and often change within inflammatory or infectious environments. This led to debates over cell identification and questioned even the mere existence of DCs as distinct leukocyte lineage. Here, we review experimental approaches taken to fate map DCs and discuss how these have shaped our understanding of DC ontogeny and lineage affiliation. Considering the ontogenetic properties of DCs will help to overcome the inherent shortcomings of purely phenotypic- and function-based approaches to cell definition and will yield a more robust way of DC classification.
RESUMEN
The duration and/or the magnitude of Ras-Erk activation are known to be crucial for cell-fate decisions. In T cells, sustained Erk activation correlates with differentiation/proliferation, whereas transient Erk activation parallels with unresponsiveness/apoptosis. The mechanism by which Son of sevenless (Sos) proteins and Ras guanyl-releasing protein 1 (RasGRP1) contribute to dynamics of Erk activation in mature T cells is not yet known. Here, we have assessed this issue using stimuli inducing either transient or sustained TCR signaling and RNA interference mediated suppression of Sos1, Sos2, and RasGRP1 expression in primary human T cells. We found that transient Erk activation depends on RasGRP1 but not on Sos. Conversely, sustained Erk signaling and T-cell activation depend on both Sos1 and RasGRP1. In summary, our data show for the first time that the two guanine nucleotide exchange factors expressed in T cells are differentially involved in the regulation of the duration of Erk phosphorylation and T-cell activation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/inmunología , Activación de Linfocitos/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Receptores de Antígenos de Linfocitos T/inmunología , Proteína SOS1/inmunología , Linfocitos T/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/genética , Femenino , Regulación de la Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/inmunología , Humanos , Masculino , Fosforilación/fisiología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína SOS1/genética , Proteína SOS1/metabolismo , Proteínas Son Of Sevenless/genética , Proteínas Son Of Sevenless/inmunología , Proteínas Son Of Sevenless/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
The lymphocyte-specific Src family protein tyrosine kinase p56(Lck) (Lck) is essential for T cell development and activation and, hence, for adaptive immune responses. The mechanism by which Lck activity is directed toward specific substrates in response to T cell receptor (TCR) activation remains elusive. We used fluorescence lifetime imaging microscopy to assess the activation-dependent spatiotemporal changes in the conformation of Lck in live human T cells. Kinetic analysis of the fluorescence lifetime of Lck biosensors enabled the direct visualization of the dynamic local opening of 20% of the total amount of Lck proteins after activation of T cells with antibody against CD3 or by superantigen-loaded antigen-presenting cells. Parallel biochemical analysis of TCR complexes revealed that the conformational changes in Lck correlated with the induction of Lck enzymatic activity. These data show the dynamic, local activation through conformational change of Lck at sites of TCR engagement.
Asunto(s)
Activación de Linfocitos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Linfocitos T/inmunología , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Microscopía Fluorescente , Conformación ProteicaRESUMEN
BACKGROUND: Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses. RESULTS: Here, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated. CONCLUSIONS: In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.
Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Anticuerpos/farmacología , Proliferación Celular , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Humanos , Proteínas Inmovilizadas/farmacología , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosforilación , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses. Here, we have analyzed signaling dynamics induced upon TCR ligation in primary human T cells. We used CD3 antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs), two widely employed methods to stimulate T cells in vitro. We show that classical sAbs stimulation induces a transient and abortive response, whereas iAbs induce sustained TCR-mediated signaling, resulting in productive T-cell responses previously observed only in antigen-specific murine systems. In summary, our analysis documents TCR signaling kinetics and suggests that iAbs are better suited for studying TCR-mediated signaling as they mimic antigen specific systems.
Asunto(s)
Anticuerpos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos/metabolismo , Anticuerpos Inmovilizados/inmunología , Anticuerpos Inmovilizados/metabolismo , Western Blotting , Complejo CD3/inmunología , Células Cultivadas , Humanos , Cinética , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Microesferas , Unión Proteica/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de la Célula Individual/métodos , Soluciones , Linfocitos T/metabolismoRESUMEN
Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sosbut not RasGRP1are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.