RESUMO
Cancer immunotherapy relies on improving T cell effector functions against malignancies, but despite the identification of several key transcription factors (TFs), the biological functions of these TFs are not entirely understood. We developed and utilized a novel, clinically relevant murine model to dissect the functional properties of crucial T cell transcription factors during anti-tumor responses. Our data showed that the loss of TCF-1 in CD8 T cells also leads to loss of key stimulatory molecules such as CD28. Our data showed that TCF-1 suppresses surface NKG2D expression on naïve and activated CD8 T cells via key transcriptional factors Eomes and T-bet. Using both in vitro and in vivo models, we uncovered how TCF-1 regulates critical molecules responsible for peripheral CD8 T cell effector functions. Finally, our unique genetic and molecular approaches suggested that TCF-1 also differentially regulates essential kinases. These kinases, including LCK, LAT, ITK, PLC-γ1, P65, ERKI/II, and JAK/STATs, are required for peripheral CD8 T cell persistent function during alloimmunity. Overall, our molecular and bioinformatics data demonstrate the mechanism by which TCF-1 modulated several critical aspects of T cell function during CD8 T cell response to cancer. Summary Figure: TCF-1 is required for persistent function of CD8 T cells but dispensable for anti-tumor response. Here, we have utilized a novel mouse model that lacks TCF-1 specifically on CD8 T cells for an allogeneic transplant model. We uncovered a molecular mechanism of how TCF-1 regulates key signaling pathways at both transcriptomic and protein levels. These key molecules included LCK, LAT, ITK, PLC-γ1, p65, ERK I/II, and JAK/STAT signaling. Next, we showed that the lack of TCF-1 impacted phenotype, proinflammatory cytokine production, chemokine expression, and T cell activation. We provided clinical evidence for how these changes impact GVHD target organs (skin, small intestine, and liver). Finally, we provided evidence that TCF-1 regulates NKG2D expression on mouse naïve and activated CD8 T cells. We have shown that CD8 T cells from TCF-1 cKO mice mediate cytolytic functions via NKG2D.
Assuntos
Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias , Fator 1 de Transcrição de Linfócitos T , Animais , Camundongos , Linfócitos T CD8-Positivos , Expressão Gênica , Neoplasias/metabolismo , Transdução de SinaisRESUMO
The transcription factor T cell factor-1 (TCF-1) is encoded by Tcf7 and plays a significant role in regulating immune responses to cancer and pathogens. TCF-1 plays a central role in CD4 T cell development; however, the biological function of TCF-1 on mature peripheral CD4 T cell-mediated alloimmunity is currently unknown. This report reveals that TCF-1 is critical for mature CD4 T cell stemness and their persistence functions. Our data show that mature CD4 T cells from TCF-1 cKO mice did not cause graft versus host disease (GvHD) during allogeneic CD4 T cell transplantation, and donor CD4 T cells did not cause GvHD damage to target organs. For the first time, we showed that TCF-1 regulates CD4 T cell stemness by regulating CD28 expression, which is required for CD4 stemness. Our data showed that TCF-1 regulates CD4 effector and central memory formation. For the first time, we provide evidence that TCF-1 differentially regulates key chemokine and cytokine receptors critical for CD4 T cell migration and inflammation during alloimmunity. Our transcriptomic data uncovered that TCF-1 regulates critical pathways during normal state and alloimmunity. Knowledge acquired from these discoveries will enable us to develop a target-specific approach for treating CD4 T cell-mediated diseases.
Assuntos
Linfócitos T CD4-Positivos , Doença Enxerto-Hospedeiro , Animais , Camundongos , Antígenos CD28/metabolismo , Camundongos Endogâmicos C57BL , Fatores de Transcrição/metabolismo , Transplante HomólogoRESUMO
To perform a multicenter assessment of the CT Pneumonia Analysis prototype for predicting disease severity and patient outcome in COVID-19 pneumonia both without and with integration of clinical information. Our IRB-approved observational study included consecutive 241 adult patients (> 18 years; 105 females; 136 males) with RT-PCR-positive COVID-19 pneumonia who underwent non-contrast chest CT at one of the two tertiary care hospitals (site A: Massachusetts General Hospital, USA; site B: Firoozgar Hospital Iran). We recorded patient age, gender, comorbid conditions, laboratory values, intensive care unit (ICU) admission, mechanical ventilation, and final outcome (recovery or death). Two thoracic radiologists reviewed all chest CTs to record type, extent of pulmonary opacities based on the percentage of lobe involved, and severity of respiratory motion artifacts. Thin-section CT images were processed with the prototype (Siemens Healthineers) to obtain quantitative features including lung volumes, volume and percentage of all-type and high-attenuation opacities (≥ -200 HU), and mean HU and standard deviation of opacities within a given lung region. These values are estimated for the total combined lung volume, and separately for each lung and each lung lobe. Multivariable analyses of variance (MANOVA) and multiple logistic regression were performed for data analyses. About 26% of chest CTs (62/241) had moderate to severe motion artifacts. There were no significant differences in the AUCs of quantitative features for predicting disease severity with and without motion artifacts (AUC 0.94-0.97) as well as for predicting patient outcome (AUC 0.7-0.77) (p > 0.5). Combination of the volume of all-attenuation opacities and the percentage of high-attenuation opacities (AUC 0.76-0.82, 95% confidence interval (CI) 0.73-0.82) had higher AUC for predicting ICU admission than the subjective severity scores (AUC 0.69-0.77, 95% CI 0.69-0.81). Despite a high frequency of motion artifacts, quantitative features of pulmonary opacities from chest CT can help differentiate patients with favorable and adverse outcomes.
Assuntos
COVID-19 , Adulto , Feminino , Humanos , Pulmão/diagnóstico por imagem , Masculino , Prognóstico , Estudos Retrospectivos , SARS-CoV-2 , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: This study aimed to assess if computed tomography (CT) radiomics can predict the severity and outcome of patients with coronavirus disease 2019 (COVID-19) pneumonia. METHODS: This institutional ethical board-approved study included 92 patients (mean age, 59 ± 17 years; 57 men, 35 women) with positive reverse transcription polymerase chain reaction assay for COVID-19 infection who underwent noncontrast chest CT. Two radiologists evaluated all chest CT examinations and recorded opacity type, distribution, and extent of lobar involvement. Information on symptom duration before hospital admission, the period of hospital admission, presence of comorbid conditions, laboratory data, and outcomes (recovery or death) was obtained from the medical records. The entire lung volume was segmented on thin-section Digital Imaging and Communication in Medicine images to derive whole-lung radiomics. Data were analyzed using multiple logistic regression with receiver operator characteristic area under the curve (AUC) as the output. RESULTS: Computed tomography radiomics (AUC, 0.99) outperformed clinical variables (AUC, 0.89) for prediction of the extent of pulmonary opacities related to COVID-19 pneumonia. Type of pulmonary opacities could be predicted with CT radiomics (AUC, 0.77) but not with clinical or laboratory data (AUC, <0.56; P > 0.05). Prediction of patient outcome with radiomics (AUC, 0.85) improved to an AUC of 0.90 with the addition of clinical variables (patient age and duration of presenting symptoms before admission). Among clinical variables, the combination of peripheral capillary oxygen saturation on hospital admission, duration of symptoms, platelet counts, and patient age provided an AUC of 0.81 for predicting patient outcomes. CONCLUSIONS: Radiomics from noncontrast CT reliably predict disease severity (AUC, 0.99) and outcome (AUC, 0.85) in patients with COVID-19 pneumonia.
Assuntos
Betacoronavirus , Infecções por Coronavirus/diagnóstico , Pulmão/diagnóstico por imagem , Pneumonia Viral/diagnóstico , Tomografia Computadorizada por Raios X/métodos , COVID-19 , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , SARS-CoV-2 , Índice de Gravidade de DoençaRESUMO
In allogeneic hematopoietic stem cell transplantation (HSCT), controlling graft-versus-host disease (GVHD) while maintaining graft-versus-tumor (GVT) responses is of critical importance. Using a mouse model of allogeneic HSCT, we hereby demonstrate that NKG2D expression by CD8(+) T cells plays a major role in mediating GVHD and GVT effects by promoting the survival and cytotoxic function of CD8(+) T cells. The expression of NKG2D ligands was not induced persistently on normal tissues of allogeneic HSCT-recipient mice treated with anti-NKG2D antibody, suggesting that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8(+) T cells to regain their GVT function. Indeed, short-term treatment with anti-NKG2D antibody restored GVT effects while maintaining an attenuated GVHD state. NKG2D expression was also detected on CD8(+) T cells from allogeneic HSCT patients and trended to be higher in those with active GVHD. Together, these data support a novel role for NKG2D expression by CD8(+) T cells during allogeneic HSCT, which could be potentially therapeutically exploited to separate GVHD from GVT effects.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação da Expressão Gênica/imunologia , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Tumor/imunologia , Transplante de Células-Tronco Hematopoéticas , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Aloenxertos , Animais , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Efeito Enxerto vs Tumor/efeitos dos fármacos , Efeito Enxerto vs Tumor/genética , Camundongos , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genéticaRESUMO
Natural killer group 2, member D (NKG2D) is a stimulatory receptor expressed by NK cells and a subset of T cells. NKG2D is crucial in diverse aspects of innate and adaptive immune functions. In this study, we characterize a novel splice variant of human NKG2D that encodes a truncated receptor lacking the ligand-binding ectodomain. This truncated NKG2D (NKG2D(TR)) isoform was detected in primary human NK and CD8(+) T cells. Overexpression of NKG2D(TR) severely attenuated cell killing and IFN-γ release mediated by full-length NKG2D (NKG2D(FL)). In contrast, specific knockdown of endogenously expressed NKG2D(TR) enhanced NKG2D-mediated cytotoxicity, suggesting that NKG2D(TR) is a negative regulator of NKG2D(FL). Biochemical studies demonstrated that NKG2D(TR) was bound to DNAX-activated protein of 10 kDa (DAP10) and interfered with the interaction of DAP10 with NKG2D(FL). In addition, NKG2D(TR) associated with NKG2D(FL), which led to forced intracellular retention, resulting in decreased surface NKG2D expression. Taken together, these data suggest that competitive interference of NKG2D/DAP10 complexes by NKG2D(TR) constitutes a novel mechanism for regulation of NKG2D-mediated function in human CD8(+) T cells and NK cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Isoformas de Proteínas/genética , Receptores Imunológicos/imunologia , Processamento Alternativo/genética , Animais , Sequência de Bases , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Interferon gama/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Isoformas de Proteínas/imunologia , Interferência de RNA , RNA Interferente Pequeno , Análise de Sequência de DNARESUMO
Transcription factors play a major role in regulation and orchestration of immune responses. The immunological context of the response can alter the regulatory networks required for proper functioning. While these networks have been well-studied in canonical immune contexts like infection, the transcription factor landscape during alloactivation remains unclear. This review addresses how transcription factors contribute to the functioning of mature alloactivated T cells. This review will also examine how these factors form a regulatory network to control alloresponses, with a focus specifically on those factors expressed by and controlling activity of T cells of the various subsets involved in graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) responses.
Assuntos
Doença Enxerto-Hospedeiro , Neoplasias , Humanos , Linfócitos T , Fatores de Transcrição/genética , Transplante HomólogoRESUMO
Regulatory T cells are suppressive immune cells used in various clinical and therapeutic applications. Canonical regulatory T cells express CD4, FOXP3, and CD25, which are considered definitive markers of their regulatory T-cell status when expressed together. However, a subset of noncanonical regulatory T cells expressing only CD4 and FOXP3 have recently been described in some infection contexts. Using a unique mouse model for the first time demonstrated that the TCF-1 regulation of regulatory T-cell suppressive function is not limited to the thymus during development. Our data showed that TCF-1 also regulated regulatory T cells' suppressive ability in secondary organs and graft-vs-host disease target organs as well as upregulating noncanonical regulatory T cells. Our data demonstrated that TCF-1 regulates the suppressive function of regulatory T cells through critical molecules like GITR and PD-1, specifically by means of noncanonical regulatory T cells. Our in vitro approaches show that TCF-1 regulates the regulatory T-cell effector-phenotype and the molecules critical for regulatory T-cell migration to the site of inflammation. Using in vivo models, we show that both canonical and noncanonical regulatory T cells from TCF-1 cKO mice have a superior suppressive function, as shown by their ability to control conventional T-cell proliferation, avert acute graft-vs-host disease, and limit tissue damage. Thus, for the first time, we provide evidence that TCF-1 negatively regulates the suppressive ability of canonical and noncanonical regulatory T cells. These findings provide evidence that TCF-1 is a novel target for developing strategies to treat alloimmune disorders.
Assuntos
Doença Enxerto-Hospedeiro , Linfócitos T Reguladores , Animais , Camundongos , Fatores de Transcrição Forkhead/genética , Inflamação , Subunidade alfa de Receptor de Interleucina-2/genética , FenótipoRESUMO
Recent evidence suggests that a rare population of self-renewing cancer stem cells (CSC) is responsible for cancer progression and therapeutic resistance. Chronic myeloid leukemia (CML) represents an important paradigm for understanding the genetic and epigenetic events involved in CSC production. CML progresses from a chronic phase (CP) in hematopoietic stem cells (HSC) that harbor the BCR-ABL translocation, to blast crisis (BC), characterized by aberrant activation of beta-catenin within granulocyte-macrophage progenitors (GMP). A major barrier to predicting and inhibiting blast crisis transformation has been the identification of mechanisms driving beta-catenin activation. Here we show that BC CML myeloid progenitors, in particular GMP, serially transplant leukemia in immunocompromised mice and thus are enriched for leukemia stem cells (LSC). Notably, cDNA sequencing of Wnt/beta-catenin pathway regulatory genes, including adenomatous polyposis coli, GSK3beta, axin 1, beta-catenin, lymphoid enhancer factor-1, cyclin D1, and c-myc, revealed a novel in-frame splice deletion of the GSK3beta kinase domain in the GMP of BC samples that was not detectable by sequencing in blasts or normal progenitors. Moreover, BC CML progenitors with misspliced GSK3beta have enhanced beta-catenin expression as well as serial engraftment potential while reintroduction of full-length GSK3beta reduces both in vitro replating and leukemic engraftment. We propose that CP CML is initiated by BCR-ABL expression in an HSC clone but that progression to BC may include missplicing of GSK3beta in GMP LSC, enabling unphosphorylated beta-catenin to participate in LSC self-renewal. Missplicing of GSK3beta represents a unique mechanism for the emergence of BC CML LSC and might provide a novel diagnostic and therapeutic target.
Assuntos
Processamento Alternativo/genética , Quinase 3 da Glicogênio Sintase/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/enzimologia , Animais , Sequência de Bases , Crise Blástica/enzimologia , Crise Blástica/patologia , Glicogênio Sintase Quinase 3 beta , Células Progenitoras de Granulócitos e Macrófagos/patologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Camundongos , Dados de Sequência Molecular , Transplante de Células-TroncoRESUMO
The focus of this review is to examine the role of ITK signaling in multiple diseases and investigate the clinical potential of ITK inhibition. The diseases and potential interventions reviewed include T cell-derived malignancies as well as other neoplastic diseases, allergic diseases such as asthma and atopic dermatitis, certain infectious diseases, several autoimmune disorders such as rheumatoid arthritis and psoriasis, and finally the use of ITK inhibition in both solid organ and bone marrow transplantation recipients.
RESUMO
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most widely applied forms of adoptive immunotherapy for the treatment of hematological malignancies. Detrimental graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia (GVL) effects occurring after allo-HSCT are largely mediated by alloantigen-reactive donor T cells in the graft. Separating GVHD from GVL effects is a formidable challenge, and a greater understanding of donor T cell biology is required to accomplish the uncoupling of GVHD from GVL. Here, we evaluated the role of ß-catenin in this process. Using a unique mouse model of transgenic overexpression of human ß-catenin (Cat-Tg) in an allo-HSCT model, we show here that T cells from Cat-Tg mice did not cause GVHD, and surprisingly, Cat-Tg T cells maintained the GVL effect. Donor T cells from Cat-Tg mice exhibited significantly lower inflammatory cytokine production and reduced donor T cell proliferation, while upregulating cytotoxic mediators that resulted in enhanced cytotoxicity. RNA sequencing revealed changes in the expression of 1169 genes for CD4, and 1006 genes for CD8+ T cells involved in essential aspects of immune response and GVHD pathophysiology. Altogether, our data suggest that ß-catenin is a druggable target for developing therapeutic strategies to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.
RESUMO
Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2-inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of "noncanonical" CD4+ CD25- FOXP3+ Tregs (ncTregs), as well as of "canonical" CD4+ CD25+ FOXP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft-versus-host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft-versus-leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk-/- ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders.
Assuntos
Interleucina-2/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Eritrócitos/metabolismo , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos C57BL/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/fisiologiaRESUMO
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also mediated by these cells. Here, we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in GVL vs. GVHD effects after allo-HSCT. CD8+ and CD4+ donor T cells from mice expressing a Y145F mutation in SLP-76 did not cause GVHD but preserved GVL effects against B-ALL cells. SLP76Y145FKI CD8+ and CD4+ donor T cells also showed less inflammatory cytokine production and migration to GVHD target organs. We developed a novel peptide to specifically inhibit SLP76:ITK interactions, resulting in decreased phosphorylation of PLCγ1 and ERK, decreased cytokine production in human T cells, and separation of GVHD from GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to separate GVHD from GVL effects after allo-HSCT treatment.
RESUMO
PURPOSE: Optimization of CT scan practices can help achieve and maintain optimal radiation protection. The aim was to assess centering, scan length, and positioning of patients undergoing chest CT for suspected or known COVID-19 pneumonia and to investigate their effect on associated radiation doses. METHODS: With respective approvals from institutional review boards, we compiled CT imaging and radiation dose data from four hospitals belonging to four countries (Brazil, Iran, Italy, and USA) on 400 adult patients who underwent chest CT for suspected or known COVID-19 pneumonia between April 2020 and August 2020. We recorded patient demographics and volume CT dose index (CTDIvol) and dose length product (DLP). From thin-section CT images of each patient, we estimated the scan length and recorded the first and last vertebral bodies at the scan start and end locations. Patient mis-centering and arm position were recorded. Data were analyzed with analysis of variance (ANOVA). RESULTS: The extent and frequency of patient mis-centering did not differ across the four CT facilities (>0.09). The frequency of patients scanned with arms by their side (11-40% relative to those with arms up) had greater mis-centering and higher CTDIvol and DLP at 2/4 facilities (p = 0.027-0.05). Despite lack of variations in effective diameters (p = 0.14), there were significantly variations in scan lengths, CTDIvol and DLP across the four facilities (p < 0.001). CONCLUSIONS: Mis-centering, over-scanning, and arms by the side are frequent issues with use of chest CT in COVID-19 pneumonia and are associated with higher radiation doses.
Assuntos
COVID-19 , Proteção Radiológica , Adulto , Braço , Humanos , Irã (Geográfico) , Itália/epidemiologia , Pandemias , Doses de Radiação , SARS-CoV-2RESUMO
Cytokine-induced killer (CIK) cells are ex vivo-expanded T lymphocytes expressing both natural killer (NK)- and T-cell markers. CIK cells are cytotoxic against autologous and allogeneic tumors. We previously showed that adoptive transfer of allogeneic CIK cells in a murine model caused minimal graft-versus-host disease (GVHD). However, the precise mechanism of reduced GVHD is not fully understood. Therefore, we evaluated the trafficking and survival of luciferase-expressing CIK cells in an allogeneic bone marrow transplant model. The initial trafficking patterns of CIK cells were similar to conventional T cells that induced GVHD; however, CIK cells infiltrated GVHD target tissues much less and transiently. CIK cells accumulated and persisted in tumor sites, resulting in tumor eradication. We evaluated different properties of CIK cells compared with conventional T cells, demonstrating a slower division rate of CIK cells, higher susceptibility to apoptosis, persistent increased expression of interferon gamma (IFN-gamma), and reduced acquisition of homing molecules required for entry of cells into inflamed GVHD target organs that lack expression of NKG2D ligands recognized by CIK cells. Due to these properties, allogeneic CIK cells had reduced expansion and caused less tissue damage. We conclude that CIK cells have the potential to separate graft-versus-tumor effects from GVHD.
Assuntos
Transferência Adotiva/métodos , Quimiotaxia de Leucócito , Citotoxicidade Imunológica , Doença Enxerto-Hospedeiro/prevenção & controle , Células Matadoras Naturais/transplante , Linfócitos T/transplante , Transferência Adotiva/normas , Animais , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citocinas/farmacologia , Doença Enxerto-Hospedeiro/etiologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos , Neoplasias/terapia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The success of cancer therapies based on allogeneic hematopoietic stem cell transplant relies on the ability to separate graft-versus-host disease (GvHD) from graft-versus-tumor (GVT) responses. Controlling donor T cell migration into peripheral tissues is a viable option to limit unwanted tissue damage, but a lack of specific targets limits progress on this front. Here, we show that the adaptor protein CrkL, but not the closely related family members CrkI or CrkII, is a crucial regulator of T cell migration. In vitro, CrkL-deficient T cells fail to polymerize actin in response to the integrin ligand ICAM-1, resulting in defective migration. Using a mouse model of GvHD/GVT, we found that while CrkL-deficient T cells can efficiently eliminate hematopoietic tumors they are unable to migrate into inflamed organs, such as the liver and small intestine, and thus do not cause GvHD. These results suggest a specific role for CrkL in trafficking to peripheral organs but not the lymphatic system. In line with this, we found that although CrkL-deficient T cells could clear hematopoietic tumors, they failed to clear the same tumor growing subcutaneously, highlighting the role of CrkL in controlling T cell migration into peripheral tissues. Our results define a unique role for CrkL in controlling T cell migration, and suggest that CrkL function could be therapeutically targeted to enhance the efficacy of immunotherapies involving allogeneic donor cells.
RESUMO
Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8+ and CD4+ donor T cells from Itk-/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk-/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.
Assuntos
Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Efeito Enxerto vs Leucemia/genética , Efeito Enxerto vs Leucemia/imunologia , Transplante de Células-Tronco Hematopoéticas , Proteínas Tirosina Quinases/genética , Animais , Movimento Celular/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Diagnóstico Diferencial , Modelos Animais de Doenças , Regulação da Expressão Gênica , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Imunidade Inata , Memória Imunológica , Imunomodulação , Interleucina-2/metabolismo , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante HomólogoRESUMO
The new coronavirus disease 2019 (COVID-19) pandemic has challenged many healthcare systems around the world. While most of the current understanding of the clinical features of COVID-19 is derived from Chinese studies, there is a relative paucity of reports from the remaining global health community. In this study, we analyze the clinical and radiologic factors that correlate with mortality odds in COVID-19 positive patients from a tertiary care center in Tehran, Iran. A retrospective cohort study of 90 patients with reverse transcriptase-polymerase chain reaction (RT-PCR) positive COVID-19 infection was conducted, analyzing demographics, co-morbidities, presenting symptoms, vital signs, laboratory values, chest radiograph findings, and chest CT features based on mortality. Chest radiograph was assessed using the Radiographic Assessment of Lung Edema (RALE) scoring system. Chest CTs were assessed according to the opacification pattern, distribution, and standardized severity score. Initial and follow-up Chest CTs were compared if available. Multiple logistic regression was used to generate a prediction model for mortality. The 90 patients included 59 men and 31 women (59.4 ± 16.6 years), including 21 deceased and 69 surviving patients. Among clinical features, advanced age (p = 0.02), low oxygenation saturation (p<0.001), leukocytosis (p = 0.02), low lymphocyte fraction (p = 0.03), and low platelet count (p = 0.048) were associated with increased mortality. High RALE score on initial chest radiograph (p = 0.002), presence of pleural effusions on initial CT chest (p = 0.005), development of pleural effusions on follow-up CT chest (p = 0.04), and worsening lung severity score on follow-up CT Chest (p = 0.03) were associated with mortality. A two-factor logistic model using patient age and oxygen saturation was created, which demonstrates 89% accuracy and area under the ROC curve of 0.86 (p<0.0001). Specific demographic, clinical, and imaging features are associated with increased mortality in COVID-19 infections. Attention to these features can help optimize patient management.
Assuntos
Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/mortalidade , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/mortalidade , Adulto , Idoso , Betacoronavirus , COVID-19 , Comorbidade , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Irã (Geográfico) , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Pandemias , Radiografia Torácica , Estudos Retrospectivos , Fatores de Risco , SARS-CoV-2 , Índice de Gravidade de Doença , Centros de Atenção Terciária , Tomografia Computadorizada por Raios XRESUMO
Cytokine-induced killer (CIK) cells are ex vivo activated and expanded CD8+ natural killer T cells that have been shown to have antitumor activity. This is the first study exploring cell killing of primary ovarian carcinoma cells with and without bispecific antibodies. Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. Bispecific antibodies against cancer antigen-125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On fluorescence-activated cell sorting analysis, the expansion of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by bispecific antibodies, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 +/- 0.3% to 89.4 +/- 2.1% at an E:T ratio of 100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001 and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.
Assuntos
Anticorpos Biespecíficos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Ovarianas/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Citocinas/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Feminino , Humanos , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/citologia , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Modern imaging technologies that allow for in vivo monitoring of cells in intact research subjects have opened up broad new areas of investigation. In the field of hematopoiesis and stem cell research, studies of cell trafficking involved in injury repair and hematopoietic engraftment have made great progress using these new tools. Multiple imaging modalities are available, each with its own advantages and disadvantages, depending on the specific application. For mouse models, clinically validated technologies such as magnetic resonance imaging (MRI) and positron emission tomography (PET) have been joined by optical imaging techniques such as in vivo bioluminescence imaging (BLI) and fluorescence imaging, and all have been used to monitor bone marrow and stem cells after transplantation into mice. Each modality requires that the cells of interest be marked with a distinct label that makes them uniquely visible using that technology. For each modality, there are several labels to choose from. Finally, multiple methods for applying these different labels are available. This chapter provides an overview of the imaging technologies and commonly used labels for each, as well as detailed protocols for gene delivery into hematopoietic cells for the purposes of applying these labels. The goal of this chapter is to provide adequate background information to allow the design and implementation of an experimental system for in vivo imaging in mice.