RESUMEN
The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αß heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αß+ γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R-STAT5B signaling promotes a supraphysiological accumulation of CD8αß+ γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αß+ γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αß+ γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer.
Asunto(s)
Inmunidad Innata , Interferón gamma , Receptores de Antígenos de Linfocitos T gamma-delta , Receptores de Interleucina-7 , Factor de Transcripción STAT5 , Timo , Animales , Interferón gamma/metabolismo , Interferón gamma/inmunología , Ratones , Humanos , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Timo/inmunología , Receptores de Interleucina-7/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunología , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Antígenos CD8/metabolismo , Femenino , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Interleucina-7/metabolismoRESUMEN
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.
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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
Development of T cells is controlled by the signal strength of the TCR. The scaffold protein kinase D-interacting substrate of 220 kilodalton (Kidins220) binds to the TCR; however, its role in T cell development was unknown. Here, we show that T cell-specific Kidins220 knockout (T-KO) mice have strongly reduced invariant natural killer T (iNKT) cell numbers and modest decreases in conventional T cells. Enhanced apoptosis due to increased TCR signaling in T-KO iNKT thymocytes of developmental stages 2 and 3 shows that Kidins220 down-regulates TCR signaling at these stages. scRNA-seq indicated that the transcription factor Aiolos is down-regulated in Kidins220-deficient iNKT cells. Analysis of an Aiolos KO demonstrated that Aiolos is a downstream effector of Kidins220 during iNKT cell development. In the periphery, T-KO iNKT cells show reduced TCR signaling upon stimulation with α-galactosylceramide, suggesting that Kidins220 promotes TCR signaling in peripheral iNKT cells. Thus, Kidins220 reduces or promotes signaling dependent on the iNKT cell developmental stage.
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Factor de Transcripción Ikaros , Proteínas de la Membrana , Células T Asesinas Naturales , Timo , Animales , Ratones , Diferenciación Celular , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Proteínas de la Membrana/metabolismo , Factor de Transcripción Ikaros/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21R110) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.
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Leucemia Linfocítica Crónica de Células B , Humanos , Femenino , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/terapia , Linfocitos B , Mutación , Receptores de Antígenos de Linfocitos B/genética , Linfocitos TRESUMEN
The ratio between κ and λ light chain (LC)-expressing B cells varies considerably between species. We recently identified Kinase D-interacting substrate of 220 kDa (Kidins220) as an interaction partner of the BCR. In vivo ablation of Kidins220 in B cells resulted in a marked reduction of λLC-expressing B cells. Kidins220 knockout B cells fail to open and recombine the genes of the Igl locus, even in genetic scenarios where the Igk genes cannot be rearranged or where the κLC confers autoreactivity. Igk gene recombination and expression in Kidins220-deficient B cells is normal. Kidins220 regulates the development of λLC B cells by enhancing the survival of developing B cells and thereby extending the time-window in which the Igl locus opens and the genes are rearranged and transcribed. Further, our data suggest that Kidins220 guarantees optimal pre-BCR and BCR signaling to induce Igl locus opening and gene recombination during B cell development and receptor editing.
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Linfocitos B , Transducción de Señal , Linfocitos B/metabolismoRESUMEN
Few cancers can be targeted efficiently by engineered T cell strategies. Here, we show that γδ T cell antigen receptor (γδ TCR)-mediated cancer metabolome targeting can be combined with targeting of cancer-associated stress antigens (such as NKG2D ligands or CD277) through the addition of chimeric co-receptors. This strategy overcomes suboptimal γ9δ2 TCR engagement of αß T cells engineered to express a defined γδ TCR (TEGs) and improves serial killing, proliferation and persistence of TEGs. In vivo, the NKG2D-CD28WT chimera enabled control only of liquid tumors, whereas the NKG2D-4-1BBCD28TM chimera prolonged persistence of TEGs and improved control of liquid and solid tumors. The CD277-targeting chimera (103-4-1BB) was the most optimal co-stimulation format, eradicating both liquid and solid tumors. Single-cell transcriptomic analysis revealed that NKG2D-4-1BBCD28TM and 103-4-1BB chimeras reprogram TEGs through NF-κB. Owing to competition with naturally expressed NKG2D in CD8+ TEGs, the NKG2D-4-1BBCD28TM chimera mainly skewed CD4+ TEGs toward adhesion, proliferation, cytotoxicity and less exhausted signatures, whereas the 103-4-1BB chimera additionally shaped the CD8+ subset toward a proliferative state.
Asunto(s)
Neoplasias , Linfocitos T , Humanos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Perfilación de la Expresión GénicaRESUMEN
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
We have developed a new format of a chimeric antigen receptor for αß T cells, in which the single-chain variable fragment recognizing the tumor antigen is directly fused to the T cell receptor, called T cell receptor fusion construct (TRuC). Here, we express an anti-CD19 εTRuC in primary γδ T cells that were expanded using zoledronate (Zol) or concanavalin A. We show that the resulting εTRuC γδ T cells were reprogrammed to better recognize CD19-positive B cell tumors and-in case of the Zol-expanded cells-a CD19-expressing colon adenocarcinoma-derived cell line in vitro. This resulted in enhanced tumor killing, upregulation of the activation marker CD25, and secretion of cytokines. We found that the transduction efficiency of the concanavalin A-expanded cells was better than the one of the Zol-expanded ones. Our in vitro cytotoxicity data suggest that the Vδ2 T cells were better killers than the Vδ1 T cells. Finally, addition of vitamin C promoted the recovery of larger γδ T cell numbers after lentiviral transduction, as used for the expression of the εTRuC. In conclusion, the generation and use of γδ εTRuC T cells might be a new approach for cancer immunotherapy.
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Adenocarcinoma , Neoplasias del Colon , Humanos , Receptores de Antígenos de Linfocitos T gamma-delta , Concanavalina A , Neoplasias del Colon/terapia , Ácido Zoledrónico/farmacología , Antígenos CD19RESUMEN
Mutations affecting T-cell receptor (TCR) signaling typically cause combined immunodeficiency (CID) due to varying degrees of disturbed T-cell homeostasis and differentiation. Here, we describe two cousins with CID due to a novel nonsense mutation in LCK and investigate the effect of this novel nonsense mutation on TCR signaling, T-cell function, and differentiation. Patients underwent clinical, genetic, and immunological investigations. The effect was addressed in primary cells and LCK-deficient T-cell lines after expression of mutated LCK. RESULTS: Both patients primarily presented with infections in early infancy. The LCK mutation led to reduced expression of a truncated LCK protein lacking a substantial part of the kinase domain and two critical regulatory tyrosine residues. T cells were oligoclonal, and especially naïve CD4 and CD8 T-cell counts were reduced, but regulatory and memory including circulating follicular helper T cells were less severely affected. A diagnostic hallmark of this immunodeficiency is the reduced surface expression of CD4. Despite severely impaired TCR signaling mTOR activation was partially preserved in patients' T cells. LCK-deficient T-cell lines reconstituted with mutant LCK corroborated partially preserved signaling. Despite detectable differentiation of memory and effector T cells, their function was severely disturbed. NK cell cytotoxicity was unaffected. Residual TCR signaling in LCK deficiency allows for reduced, but detectable T-cell differentiation, while T-cell function is severely disturbed. Our findings expand the previous report on one single patient on the central role of LCK in human T-cell development and function.
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Síndromes de Inmunodeficiencia , Enfermedades de Inmunodeficiencia Primaria , Humanos , Codón sin Sentido , Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosforilación , Enfermedades de Inmunodeficiencia Primaria/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cells harbor the T cell receptor (TCR)-derived ζ chain as an intracellular activation domain in addition to costimulatory domains. The functionality in a CAR format of the other chains of the TCR complex, namely CD3δ, CD3ε and CD3γ, instead of ζ, remains unknown. In the present study, we have systematically engineered new CD3 CARs, each containing only one of the CD3 intracellular domains. We found that CARs containing CD3δ, CD3ε or CD3γ cytoplasmic tails outperformed the conventional ζ CAR T cells in vivo. Transcriptomic and proteomic analysis revealed differences in activation potential, metabolism and stimulation-induced T cell dysfunctionality that mechanistically explain the enhanced anti-tumor performance. Furthermore, dimerization of the CARs improved their overall functionality. Using these CARs as minimalistic and synthetic surrogate TCRs, we have identified the phosphatase SHP-1 as a new interaction partner of CD3δ that binds the CD3δ-ITAM on phosphorylation of its C-terminal tyrosine. SHP-1 attenuates and restrains activation signals and might thus prevent exhaustion and dysfunction. These new insights into T cell activation could promote the rational redesign of synthetic antigen receptors to improve cancer immunotherapy.
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Proteómica , Receptores de Antígenos de Linfocitos T , Complejo CD3 , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Membrana Celular/metabolismo , Activación de Linfocitos , Linfocitos TRESUMEN
There are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γδ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γδ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γδ T cells. Indeed, neither promigratory engineered γδ T cells, nor anti-PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γδ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.
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Receptores de Antígenos de Linfocitos T gamma-delta , Neoplasias de la Mama Triple Negativas , Humanos , Ratones , Animales , Neoplasias de la Mama Triple Negativas/metabolismo , Monitorización Inmunológica , Recurrencia Local de Neoplasia/patología , Células Madre NeoplásicasRESUMEN
The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction-mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor-treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. IMPLICATIONS: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II-production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti-PD-1 immunotherapy for metastatic melanoma is planned.
Asunto(s)
Antineoplásicos , Melanoma , Animales , Ratones , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Interleucina-15/metabolismo , Interleucina-15/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Línea Celular Tumoral , Melanoma/tratamiento farmacológico , Melanoma/genética , Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Microambiente TumoralRESUMEN
The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.
RESUMEN
Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human ß-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal ß-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal ß-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.
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Enfermedad Injerto contra Huésped , beta-Defensinas , Humanos , Animales , Ratones , beta-Defensinas/genética , beta-Defensinas/metabolismo , beta-Defensinas/farmacología , Infiltración Neutrófila , Íleon , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/genética , Receptores de Antígenos de Linfocitos TRESUMEN
Alterations in both the expression and function of the non-receptor tyrosine kinase Zap70 are associated with numerous human diseases including immunodeficiency, autoimmunity, and leukemia. Zap70 propagates the TCR signal by phosphorylating two important adaptor molecules, LAT and SLP76, which orchestrate the assembly of the signaling complex, leading to the activation of PLCγ1 and further downstream pathways. These events are crucial to drive T-cell development and T-cell activation. Recently, it has been proposed that C564, located in the kinase domain of Zap70, is palmitoylated. A non-palmitoylable C564R Zap70 mutant, which has been reported in a patient suffering from immunodeficiency, is incapable of propagating TCR signaling and activating T cells. The lack of palmitoylation was suggested as the cause of this human disease. Here, we confirm that Zap70C564R is signaling defective, but surprisingly, the defective Zap70 function does not appear to be due to a loss in palmitoylation. We engineered a C564A mutant of Zap70 which, similarly to Zap70C564R, is non-palmitoylatable. However, this mutant was capable of propagating TCR signaling. Moreover, Zap70C564A enhanced the activity of Lck and increased its proximity to the TCR. Accordingly, Zap70-deficient P116 T cells expressing Zap70C564A displayed the hyperphosphorylation of TCR-ζ and Zap70 (Y319), two well-known Lck substrates. Collectively, these data indicate that C564 is important for the regulation of Lck activity and proximal TCR signaling, but not for the palmitoylation of Zap70.
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Cisteína , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Proteína Tirosina Quinasa ZAP-70 , Cisteína/metabolismo , Humanos , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.
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Linfoma de Burkitt , Neoplasias Colorrectales , Receptores Quiméricos de Antígenos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/terapia , Humanos , Lectinas/metabolismo , Polisacáridos/metabolismo , Linfocitos TRESUMEN
The human T-cell leukemia virus type 1 (HTLV-1) is the cause of serious malignant and inflammatory diseases, including adult T-cell leukemia and lymphoma and tropical spastic paraparesis. The potential protective role of γδ T cells in HTLV-1 infection remains unclear. Here, demonstrate that there is a decrease in the amount of Vγ9Vδ2 T cells in patients with HTLV-1, especially in those with HTLV-1 associated pathologies. This suggests that γδ T cells could be involved in controlling the virus. Indeed, we found that Vγ9Vδ2 T cells, expanded from non-infected individuals, can kill cells expressing the viral proteins HBZ and Tax and this phenotype is reversed in the presence of mevastatin. Cytotoxicity by Vγ9Vδ2 T cells was not associated with an increase of INF-γ production. In sharp contrast, killing by NK cells was reduced by Tax expression. Thus, our study provides initial evidence for a potential protective role of Vγ9Vδ2 T cells against HTLV-1 infection. Therapeutic exploitation of these insights is feasible with current technologies of T-cell therapies and could provide novel tools to prevent and treat HTLV-1-associated malignancies and neurologic complications.
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Infecciones por HTLV-I , Virus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical , Adulto , Humanos , Fenotipo , Linfocitos TRESUMEN
Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid-GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs.
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Células Madre Hematopoyéticas , Ácido Hialurónico , Animales , Médula Ósea , Hematopoyesis , Humanos , Ratones , Transducción de SeñalRESUMEN
B-cell tolerance to self-antigen is an active process that requires the temporal and spatial integration of signals of defined intensity. In common variable immune deficiency disorders, CTLA-4 deficiency, autoimmune lymphoproliferative syndrome, or in collagen VII deficiency, genetic defects in molecules regulating development, activation, maturation, and ECM composition alter the generation of B cells, resulting in immunodeficiency. Paradoxically, at the same time, the defective immune processes favor autoantibody production and immunopathology through impaired establishment of tolerance. The development of systemic autoimmunity in the framework of defective BCR signaling is relatively unusual in genetic mouse models. In sharp contrast, such reduced signaling in humans is clearly linked to pathological autoimmunity. The molecular mechanisms by which tolerance is broken in these settings are only starting to be explored resulting in novel therapeutic interventions. For instance, in CTLA-4 deficiency, homeostasis can be restored by CTLA-4 Ig treatment. Following this example, the identification of the molecular targets causing the reduced signals and their restoration is a visionary way to reestablish tolerance and develop novel therapeutic avenues for immunopathologies.
Asunto(s)
Autoinmunidad , Síndromes de Inmunodeficiencia , Animales , Anticuerpos , Antígeno CTLA-4 , Humanos , Tolerancia Inmunológica , RatonesRESUMEN
BACKGROUND: Pharmaceutical intervention in the CNS is hampered by the shielding function of the blood-brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery. METHODS: Barrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis. RESULTS: Isoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy. CONCLUSIONS: Combination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.