RESUMO
Despite significant progress over the last few decades in the treatment of acute myeloid leukemia (AML), there still remains a major unmet medical need for this disease. Immunotherapy approaches for redirecting pan CD3+ T cells to target leukemia blasts have shown limited efficacy in clinical trials and often accompanied with severe toxicity in AML patients. We designed an alternative engager molecule (Anti-TRGV9/anti-CD123), a bispecific antibody that can simultaneously bind to the Vγ9 chain of the Vγ9Vδ2+ γδ T cell receptor and to AML target antigen, CD123, to selectively recruit Vγ9+ γδ T cells rather than pan T cells to target AML blasts. Our results suggest that prototypic bispecific antibodies (a) selectively activate Vγ9+ γδ T cells as judged by CD69 and CD25 surface expression, and intracellular Granzyme B expression, (b) selectively recruit Vγ9+ γδ T cells into cell-cell conjugate formation of γδ T cells with tumor cells indicating selective and effective engagement of effector and target tumor cells, and (c) mediate γδ T cell cytotoxicity (in vitro and in vivo) against tumor antigen-expressing cells. Collectively, these findings suggest that selectively redirecting Vγ9+ γδ T cells to target AML blasts has a potential for immunotherapy for AML patients and favors further exploration of this concept.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos Imunológicos/farmacologia , Imunoterapia/métodos , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Animais , Citotoxicidade Imunológica , Humanos , Leucemia Experimental/imunologia , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Manipulating molecules that impact T cell receptor (TCR) or cytokine signaling, such as the protein tyrosine phosphatase non-receptor type 2 (PTPN2), has significant potential for advancing T cell-based immunotherapies. Nonetheless, it remains unclear how PTPN2 impacts the activation, survival, and memory formation of T cells. We find that PTPN2 deficiency renders cells in vivo and in vitro less dependent on survival-promoting cytokines, such as interleukin (IL)-2 and IL-15. Remarkably, briefly ex vivo-activated PTPN2-deficient T cells accumulate in 3- to 11-fold higher numbers following transfer into unmanipulated, antigen-free mice. Moreover, the absence of PTPN2 augments the survival of short-lived effector T cells and allows them to robustly re-expand upon secondary challenge. Importantly, we find no evidence for impaired effector function or memory formation. Mechanistically, PTPN2 deficiency causes broad changes in the expression and phosphorylation of T cell expansion and survival-associated proteins. Altogether, our data underline the therapeutic potential of targeting PTPN2 in T cell-based therapies to augment the number and survival capacity of antigen-specific T cells.
Assuntos
Ativação Linfocitária/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Linfócitos T/metabolismo , Animais , Proteínas de Transporte/metabolismo , Comunicação Celular , Citocinas/metabolismo , Feminino , Imunoterapia Adotiva/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de SinaisRESUMO
Checkpoint blockade mediates a proliferative response of tumor-infiltrating CD8+ T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1+PD-1+ TILs mediated the proliferative response to immunotherapy, generating both Tcf1+PD-1+ and differentiated Tcf1-PD-1+ cells. Ablation of Tcf1+PD-1+ TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1+PD-1+ TILs but was essential for the stem-like functions of these cells. Human TCF1+PD-1+ cells were detected among tumor-reactive CD8+ T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Melanoma/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Células-Tronco/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Assuntos
Embrião de Mamíferos/metabolismo , Eritropoese , Fator de Transcrição GATA1/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Animais , Embrião de Mamíferos/citologia , Fator de Transcrição GATA1/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Células K562 , Camundongos , Camundongos Knockout , Proteínas/genética , Subunidades Ribossômicas Maiores/genética , Subunidades Ribossômicas Maiores/metabolismoRESUMO
Although Notch signaling plays important roles in lineage commitment and differentiation of multiple cell types including conventional T cells, nothing is currently known concerning Notch function in innate-like T cells. We have found that the homeostasis of several well-characterized populations of innate-like T cells including invariant NKT cells (iNKT), CD8ααTCRαß small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells is controlled by Notch. Notch selectively regulates hepatic iNKT cell survival via tissue-restricted control of B cell lymphoma 2 and IL-7Rα expression. More generally, Notch regulation of innate-like T cell homeostasis involves both cell-intrinsic and -extrinsic mechanisms and relies upon context-dependent interactions with Notch ligand-expressing fibroblastic stromal cells. Collectively, using conditional ablation of Notch receptors on peripheral T cells or Notch ligands on putative fibroblastic stromal cells, we show that Notch signaling is indispensable for the homeostasis of three tissue-restricted populations of innate-like T cells: hepatic iNKT, CD8ααTCRαß small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells, thus supporting a generalized role for Notch in innate T cell homeostasis.
Assuntos
Diferenciação Celular/imunologia , Homeostase/imunologia , Receptores Notch/imunologia , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Receptores Notch/metabolismoRESUMO
NLR family apoptosis inhibitory proteins (NAIPs) belong to both the Nod-like receptor (NLR) and the inhibitor of apoptosis (IAP) families. NAIPs are known to form an inflammasome with NLRC4, but other in vivo functions remain unexplored. Using mice deficient for all NAIP paralogs (Naip1-6(Δ/Δ)), we show that NAIPs are key regulators of colorectal tumorigenesis. Naip1-6(Δ/Δ) mice developed increased colorectal tumors, in an epithelial-intrinsic manner, in a model of colitis-associated cancer. Increased tumorigenesis, however, was not driven by an exacerbated inflammatory response. Instead, Naip1-6(Δ/Δ) mice were protected from severe colitis and displayed increased antiapoptotic and proliferation-related gene expression. Naip1-6(Δ/Δ) mice also displayed increased tumorigenesis in an inflammation-independent model of colorectal cancer. Moreover, Naip1-6(Δ/Δ) mice, but not Nlrc4-null mice, displayed hyper-activation of STAT3 and failed to activate p53 18 h after carcinogen exposure. This suggests that NAIPs protect against tumor initiation in the colon by promoting the removal of carcinogen-elicited epithelium, likely in a NLRC4 inflammasome-independent manner. Collectively, we demonstrate a novel epithelial-intrinsic function of NAIPs in protecting the colonic epithelium against tumorigenesis.
Assuntos
Colite/patologia , Neoplasias do Colo/patologia , Proteína Inibidora de Apoptose Neuronal/metabolismo , Animais , Colite/genética , Colite/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Inflamassomos/genética , Inflamassomos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Inibidora de Apoptose Neuronal/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.
Assuntos
Interleucina-17/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Medula Óssea/metabolismo , Quimerismo , Homeostase/imunologia , Imunidade Inata , Interleucina-17/deficiência , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores CCR6/metabolismo , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismo , Timo/embriologia , Timo/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-ß and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.