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1.
Proc Natl Acad Sci U S A ; 115(10): E2311-E2319, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29463696

RESUMO

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage- Sca-1+ c-Kit+ CD150+ CD48- HSPC subset (LSK CD150+ CD48- cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48- cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.


Assuntos
Células Sanguíneas/imunologia , Hematopoese , Fator de Transcrição STAT3/imunologia , Animais , Células Sanguíneas/citologia , Linhagem da Célula , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/imunologia , Fator de Transcrição STAT3/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/imunologia
2.
J Immunol ; 201(5): 1389-1399, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021768

RESUMO

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. JClinInvest 111: 639-647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)-expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti-low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells' capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Leucemia Mieloide Aguda/imunologia , Mieloblastina/imunologia , Proteínas de Neoplasias/imunologia , Neutrófilos/imunologia , Adulto , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Feminino , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Neutrófilos/patologia
3.
J Biol Chem ; 292(24): 10295-10305, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28468826

RESUMO

Neutrophil elastase (NE) can be rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer, which results in cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapy target in hematological and solid tumor malignancies. The mechanism of NE uptake, however, remains unknown. Using the mass spectrometry-based approach, we identify neuropilin-1 (NRP1) as a NE receptor that mediates uptake and PR1 cross-presentation in breast cancer cells. We demonstrated that soluble NE is a specific, high-affinity ligand for NRP1 with a calculated Kd of 38.7 nm Furthermore, we showed that NRP1 binds to the RRXR motif in NE. Notably, NRP1 knockdown with interfering RNA or CRISPR-cas9 system and blocking using anti-NRP1 antibody decreased NE uptake and, subsequently, susceptibility to lysis by PR1-specific cytotoxic T cells. Expression of NRP1 in NRP1-deficient cells was sufficient to induce NE uptake. Altogether, because NRP1 is broadly expressed in tumors, our findings suggest a role for this receptor in immunotherapy strategies that target cross-presented antigens.


Assuntos
Absorção Fisiológica , Neoplasias da Mama/metabolismo , Apresentação Cruzada , Elastase de Leucócito/metabolismo , Proteínas de Neoplasias/metabolismo , Neuropilina-1/metabolismo , Motivos de Aminoácidos , Anticorpos Bloqueadores/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Humanos , Cinética , Elastase de Leucócito/química , Elastase de Leucócito/imunologia , Ligantes , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/química , Neuropilina-1/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
4.
Blood ; 125(19): 2968-73, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25712988

RESUMO

Tyrosine kinase inhibitors (TKIs) are used as a frontline therapy for BCR-ABL(+) acute lymphoblastic leukemia (ALL). However, resistance to TKI therapy arises rapidly, and its underlying molecular mechanisms are poorly understood. In this study, we identified a novel cascade of events initiated by TKIs and traversing through mesenchymal stem cells (MSCs) to leukemic cells, leading to resistance. MSCs exposed to TKIs acquired a new functional status with the expression of genes encoding for chemo-attractants, adhesion molecules, and prosurvival growth factors, and this priming enabled leukemic cells to form clusters underneath the MSCs. This cluster formation was associated with the protection of ALL cells from therapy as leukemic cells switched from BCR-ABL signaling to IL-7R/Janus kinase signaling to survive in the MSC milieu. Our findings illustrate a novel perspective in the evolution of TKI resistance and provide insights for advancing the treatment of BCR-ABL(+) ALL.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Mesenquimais/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
5.
Cancer Immunol Immunother ; 65(6): 741-51, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27129972

RESUMO

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.


Assuntos
Apresentação de Antígeno/imunologia , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Elastase de Leucócito/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
6.
Cytotherapy ; 18(8): 985-994, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27265873

RESUMO

BACKGROUND AIMS: The PR1 peptide, derived from the leukemia-associated antigens proteinase 3 and neutrophil elastase, is overexpressed on HLA-A2 in acute myeloid leukemia (AML). We developed a T-cell receptor (TCR)-like monoclonal antibody (8F4) that binds the PR1/HLA-A2 complex on the surface of AML cells, efficiently killing them in vitro and eliminating them in preclinical models. Humanized 8F4 (h8F4) with high affinity for the PR1/HLA-A2 epitope was used to construct an h8F4- chimeric antigen receptor (CAR) that was transduced into T cells to mediate anti-leukemia activity. METHODS: Human T cells were transduced to express the PR1/HLA-A2-specific CAR (h8F4-CAR-T cells) containing the scFv of h8F4 fused to the intracellular signaling endo-domain of CD3 zeta chain through the transmembrane and intracellular costimulatory domain of CD28. RESULTS: Adult human normal peripheral blood (PB) T cells were efficiently transduced with the h8F4-CAR construct and predominantly displayed an effector memory phenotype with a minor population (12%) of central memory cells in vitro. Umbilical cord blood (UCB) T cells could also be efficiently transduced with the h8F4-CAR. The PB and UCB-derived h8F4-CAR-T cells specifically recognized the PR1/HLA-A2 complex and were capable of killing leukemia cell lines and primary AML blasts in an HLA-A2-dependent manner. CONCLUSIONS: Human adult PB and UCB-derived T cells expressing a CAR derived from the TCR-like 8F4 antibody rapidly and efficiently kill AML in vitro. Our data could lead to a new treatment paradigm for AML in which targeting leukemia stem cells could transfer long-term immunity to protect against relapse.


Assuntos
Sangue Fetal , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/terapia , Leucócitos Mononucleares/metabolismo , Mieloblastina/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Epitopos/imunologia , Sangue Fetal/citologia , Sangue Fetal/imunologia , Terapia Genética , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/imunologia , Mieloblastina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/imunologia
7.
Cytotherapy ; 18(8): 995-1001, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27378343

RESUMO

BACKGROUND AIMS: PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci. METHODS: We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells. RESULTS: PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines. CONCLUSIONS: This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Sangue Fetal/citologia , Antígeno HLA-A2/imunologia , Imunoterapia Adotiva , Leucemia Mieloide/terapia , Mieloblastina/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Células Cultivadas , Sangue Fetal/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Células K562 , Leucemia Mieloide/imunologia , Contagem de Linfócitos , Mieloblastina/química , Mieloblastina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Células U937
8.
J Immunol ; 193(9): 4654-62, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25238756

RESUMO

Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific ssDNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim domain-binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2(-/-) mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. In this study, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2(-/-) mice, and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2(-/-) HSPCs from wild-type HSPCs. Finally, a tendency toward coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of the Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function.


Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Estresse Fisiológico , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Medula Óssea/metabolismo , Medula Óssea/patologia , Transplante de Medula Óssea , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica , Hematopoese/genética , Homeostase/genética , Imunofenotipagem , Camundongos , Camundongos Knockout , Fenótipo , Receptor Notch1/genética , Receptor Notch1/metabolismo
9.
J Immunol ; 189(11): 5476-84, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23105141

RESUMO

PR1 is a HLA-A2-restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 (P3) and neutrophil elastase (NE), which are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived APCs, and that NE is taken up by breast cancer cells. We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. We further show that PR1 cross-presentation renders human breast cancer and melanoma cells susceptible to killing by PR1-specific CTLs (PR1-CTL) and the anti-PR1/HLA-A2 Ab 8F4. We also show PR1-CTL in peripheral blood from patients with breast cancer and melanoma. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an Ag become susceptible to therapies that target cross-presented Ags and suggest PR1 as a broadly expressed tumor Ag.


Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/terapia , Imunoterapia , Elastase de Leucócito/imunologia , Melanoma/terapia , Mieloblastina/imunologia , Neoplasias Cutâneas/terapia , Anticorpos/farmacologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Antígenos de Neoplasias/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Apresentação Cruzada , Feminino , Antígeno HLA-A2/imunologia , Humanos , Elastase de Leucócito/química , Melanoma/imunologia , Melanoma/patologia , Terapia de Alvo Molecular , Mieloblastina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T Citotóxicos/imunologia , Células Tumorais Cultivadas
10.
Res Sq ; 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38464203

RESUMO

Hu8F4 is a T cell receptor (TCR)-like antibody with high affinity for leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is comprised of the Hu8F4 scFv, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain, and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from AML patients in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor (FcgR)-expressing cells. We generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with FcgR-expressing cells in vivo. The primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. Herein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor-binding-induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

11.
Leukemia ; 38(5): 1143-1155, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38467768

RESUMO

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.


Assuntos
Comunicação Autócrina , Células-Tronco Hematopoéticas , Interferon Tipo I , Fator de Transcrição STAT3 , Animais , Fator de Transcrição STAT3/metabolismo , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Interferon Tipo I/metabolismo , Transdução de Sinais , Camundongos Endogâmicos C57BL , Camundongos Knockout
12.
Neuro Oncol ; 26(5): 826-839, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38237157

RESUMO

BACKGROUND: Glioblastomas (GBMs) are central nervous system tumors that resist standard-of-care interventions and even immune checkpoint blockade. Myeloid cells in the tumor microenvironment can contribute to GBM progression; therefore, emerging immunotherapeutic approaches include reprogramming these cells to achieve desirable antitumor activity. Triggering receptor expressed on myeloid cells 2 (TREM2) is a myeloid signaling regulator that has been implicated in a variety of cancers and neurological diseases with contrasting functions, but its role in GBM immunopathology and progression is still under investigation. METHODS: Our reverse translational investigations leveraged single-cell RNA sequencing and cytometry of human gliomas to characterize TREM2 expression across myeloid subpopulations. Using 2 distinct murine glioma models, we examined the role of Trem2 on tumor progression and immune modulation of myeloid cells. Furthermore, we designed a method of tracking phagocytosis of glioma cells in vivo and employed in vitro assays to mechanistically understand the influence of TREM2 signaling on tumor uptake. RESULTS: We discovered that TREM2 expression does not correlate with immunosuppressive pathways, but rather showed strong a positive association with the canonical phagocytosis markers lysozyme (LYZ) and macrophage scavenger receptor (CD163) in gliomas. While Trem2 deficiency was found to be dispensable for gliomagenesis, Trem2+ myeloid cells display enhanced tumor uptake compared to Trem2- cells. Mechanistically, we demonstrate that TREM2 mediates phagocytosis via Syk signaling. CONCLUSIONS: These results indicate that TREM2 is not associated with immunosuppression in gliomas. Instead, TREM2 is an important regulator of phagocytosis that may be exploited as a potential therapeutic strategy for brain tumors.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glicoproteínas de Membrana , Fagocitose , Receptores Imunológicos , Animais , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Humanos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Microambiente Tumoral , Células Mieloides/metabolismo , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , Transdução de Sinais
13.
Blood ; 117(16): 4262-72, 2011 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-21296998

RESUMO

PR1 (VLQELNVTV) is a human leukocyte antigen-A2 (HLA-A2)-restricted leukemia-associated peptide from proteinase 3 (P3) and neutrophil elastase (NE) that is recognized by PR1-specific cytotoxic T lymphocytes that contribute to cytogenetic remission of acute myeloid leukemia (AML). We report a novel T-cell receptor (TCR)-like immunoglobulin G2a (IgG2a) antibody (8F4) with high specific binding affinity (dissociation constant [K(D)] = 9.9nM) for a combined epitope of the PR1/HLA-A2 complex. Flow cytometry and confocal microscopy of 8F4-labeled cells showed significantly higher PR1/HLA-A2 expression on AML blasts compared with normal leukocytes (P = .046). 8F4 mediated complement-dependent cytolysis of AML blasts and Lin(-)CD34(+)CD38(-) leukemia stem cells (LSCs) but not normal leukocytes (P < .005). Although PR1 expression was similar on LSCs and hematopoietic stem cells, 8F4 inhibited AML progenitor cell growth, but not normal colony-forming units from healthy donors (P < .05). This study shows that 8F4, a novel TCR-like antibody, binds to a conformational epitope of the PR1/HLA-A2 complex on the cell surface and mediates specific lysis of AML, including LSCs. Therefore, this antibody warrants further study as a novel approach to targeting leukemia-initiating cells in patients with AML.


Assuntos
Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica/imunologia , Epitopos/imunologia , Antígeno HLA-A2/imunologia , Leucemia Mieloide Aguda/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linhagem Celular , Humanos , Leucemia Mieloide Aguda/patologia , Leucócitos/imunologia , Leucócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de IgG/imunologia , Células-Tronco/imunologia
14.
bioRxiv ; 2023 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-37066184

RESUMO

Glioblastomas (GBMs) are tumors of the central nervous system that remain recalcitrant to both standard of care chemo-radiation and immunotherapies. Emerging approaches to treat GBMs include depletion or re-education of innate immune cells including microglia (MG) and macrophages (MACs). Here we show myeloid cell restricted expression of triggering receptor expressed on myeloid cells 2 (TREM2) across low- and high-grade human gliomas. TREM2 expression did not correlate with immunosuppressive pathways, but rather showed strong positive association with phagocytosis markers such as lysozyme (LYZ) and CD163 in gliomas. In line with these observations in patient tumors, Trem2-/- mice did not exhibit improved survival compared to wildtype (WT) mice when implanted with mouse glioma cell lines, unlike observations previously seen in peripheral tumor models. Gene expression profiling revealed pathways related to inflammation, adaptive immunity, and autophagy that were significantly downregulated in tumors from Trem2-/- mice compared to WT tumors. Using ZsGreen-expressing CT-2A orthotopic implants, we found higher tumor antigen engulfment in Trem2+ MACs, MG, and dendritic cells. Our data uncover TREM2 as an important immunomodulator in gliomas and inducing TREM2 mediated phagocytosis can be a potential immunotherapeutic strategy for brain tumors.

15.
bioRxiv ; 2023 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-36798265

RESUMO

STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern as Stat3 deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediated Stat3 deletion in 20% of the hematopoietic compartment. Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast to Stat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells revealed altered transcriptional responses in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient Lin-ckit+Sca1+ cells accumulated γH2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs.

16.
Nat Cancer ; 4(1): 62-80, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36585453

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is considered non-immunogenic, with trials showing its recalcitrance to PD1 and CTLA4 immune checkpoint therapies (ICTs). Here, we sought to systematically characterize the mechanisms underlying de novo ICT resistance and to identify effective therapeutic options for PDAC. We report that agonist 41BB and antagonist LAG3 ICT alone and in combination, increased survival and antitumor immunity, characterized by modulating T cell subsets with antitumor activity, increased T cell clonality and diversification, decreased immunosuppressive myeloid cells and increased antigen presentation/decreased immunosuppressive capability of myeloid cells. Translational analyses confirmed the expression of 41BB and LAG3 in human PDAC. Since single and dual ICTs were not curative, T cell-activating ICTs were combined with a CXCR1/2 inhibitor targeting immunosuppressive myeloid cells. Triple therapy resulted in durable complete responses. Given similar profiles in human PDAC and the availability of these agents for clinical testing, our findings provide a testable hypothesis for this lethal disease.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/tratamento farmacológico , Células Mieloides/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Receptores de Interleucina-8A/imunologia , Neoplasias Pancreáticas
17.
bioRxiv ; 2023 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-37808770

RESUMO

Myelodysplastic syndromes (MDS) are a group of incurable hematopoietic stem cell (HSC) neoplasms characterized by peripheral blood cytopenias and a high risk of progression to acute myeloid leukemia. MDS represent the final stage in a continuum of HSCs' genetic and functional alterations and are preceded by a premalignant phase, clonal cytopenia of undetermined significance (CCUS). Dissecting the mechanisms of CCUS maintenance may uncover therapeutic targets to delay or prevent malignant transformation. Here, we demonstrate that DNMT3A and TET2 mutations, the most frequent mutations in CCUS, induce aberrant HSCs' differentiation towards the myeloid lineage at the expense of erythropoiesis by upregulating IL-1ß-mediated inflammatory signaling and that canakinumab rescues red blood cell transfusion dependence in early-stage MDS patients with driver mutations in DNMT3A and TET2 . This study illuminates the biological landscape of CCUS and offers an unprecedented opportunity for MDS intervention during its initial phase, when expected survival is prolonged.

18.
J Exp Med ; 220(2)2023 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-36367776

RESUMO

Immune checkpoint blockade (ICB) has revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by immune-related adverse events (irAEs). Limited understanding of irAE mechanisms hampers development of approaches to mitigate their damage. To address this, we examined whether mice gained sensitivity to anti-CTLA-4 (αCTLA-4)-mediated toxicity upon disruption of gut homeostatic immunity. We found αCTLA-4 drove increased inflammation and colonic tissue damage in mice with genetic predisposition to intestinal inflammation, acute gastrointestinal infection, transplantation with a dysbiotic fecal microbiome, or dextran sodium sulfate administration. We identified an immune signature of αCTLA-4-mediated irAEs, including colonic neutrophil accumulation and systemic interleukin-6 (IL-6) release. IL-6 blockade combined with antibiotic treatment reduced intestinal damage and improved αCTLA-4 therapeutic efficacy in inflammation-prone mice. Intestinal immune signatures were validated in biopsies from patients with ICB colitis. Our work provides new preclinical models of αCTLA-4 intestinal irAEs, mechanistic insights into irAE development, and potential approaches to enhance ICB efficacy while mitigating irAEs.


Assuntos
Colite , Interleucina-6 , Camundongos , Animais , Qualidade de Vida , Colite/patologia , Imunoterapia , Inflamação
19.
Proc Natl Acad Sci U S A ; 106(43): 18333-8, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19815516

RESUMO

With age, T-cell generation from the thymus is much reduced, yet a substantial naïve T-cell pool is maintained even in aged animals, suggesting that naïve T cells either persist longer or turn over faster to maintain T-cell homeostasis. We found that with age, naïve CD4 T cells became progressively longer-lived. Their longer lifespan did not depend on recognition of self-peptide/class II. Newly generated naïve T cells derived from aged stem cells had a shorter lifespan, like that of young naïve T cells. Conversely, naïve CD4 T cells derived from middle-aged thymectomized mice were longer-lived in vivo, and their development of functional defects was accelerated. These observations suggest that naïve T cells develop their longer lifespan during their sojourn in the periphery. Increased longevity of naïve CD4 T cells correlated well with reduced expression of proapoptotic molecule Bim. We suggest that the intrinsic increase in longevity helps maintain naïve T-cell homeostasis but facilitates the development of functional defects in mice.


Assuntos
Envelhecimento , Linfócitos T CD4-Positivos/imunologia , Homeostase , Imunidade Inata , Linfócitos T/imunologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD4-Positivos/citologia , Sobrevivência Celular , Antígenos de Histocompatibilidade/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/citologia , Timectomia
20.
STAR Protoc ; 3(4): 101764, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36240061

RESUMO

Drug testing assays in hematopoietic stem and progenitor cells (HSPCs) are fundamental in biological studies of myelodysplastic syndromes (MDS) but have historically entailed a technical challenge. This protocol allows the efficient isolation of MDS HSPCs from bone marrow mononuclear cell fractions and their culturing with the support of stromal cells for improved maintenance during drug testing. Lastly, specific steps are given to quantify surviving cells and assess changes in the HSPC hierarchies. For complete details on the use and execution of this protocol, please refer to Ganan-Gomez et al. (2022).


Assuntos
Medula Óssea , Síndromes Mielodisplásicas , Humanos , Células-Tronco Hematopoéticas , Células Estromais , Células da Medula Óssea
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