RESUMO
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.
Assuntos
Imunidade Inata , Interferon gama , Receptores de Antígenos de Linfócitos T gama-delta , Receptores de Interleucina-7 , Fator de Transcrição STAT5 , Timo , Animais , Interferon gama/metabolismo , Interferon gama/imunologia , Camundongos , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Timo/imunologia , Receptores de Interleucina-7/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/imunologia , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos/imunologia , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Antígenos CD8/metabolismo , Feminino , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/metabolismo , Interleucina-7/metabolismoRESUMO
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.
Assuntos
Síndromes de Imunodeficiência , Doenças da Imunodeficiência Primária , Humanos , Códon sem Sentido , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Fosforilação , Doenças da Imunodeficiência Primária/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Complexo CD3 , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos , Transdução de Sinais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo CD3/metabolismo , Células HEK293 , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Células Jurkat , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Fosforilação , Ligação Proteica , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteína-Tirosina Quinase ZAP-70/genéticaRESUMO
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.
Assuntos
Adenocarcinoma , Neoplasias do Colo , Humanos , Receptores de Antígenos de Linfócitos T gama-delta , Concanavalina A , Neoplasias do Colo/terapia , Ácido Zoledrônico/farmacologia , Antígenos CD19RESUMO
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.
Assuntos
Fator de Transcrição Ikaros , Proteínas de Membrana , Células T Matadoras Naturais , Timo , Animais , Camundongos , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas de Membrana/metabolismo , Fator de Transcrição Ikaros/metabolismo , Timo/citologia , Timo/metabolismoRESUMO
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.
Assuntos
Linfócitos B , Transdução de Sinais , Linfócitos B/metabolismoRESUMO
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.
Assuntos
Síndromes de Imunodeficiência , Linfopenia , Lactente , Humanos , Animais , Camundongos , Antígenos CD28 , Linfócitos T CD4-Positivos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Receptores de Antígenos de Linfócitos T/genética , Inflamação/genética , Linfopenia/genéticaRESUMO
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.