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The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.
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Antígenos CD28 , Redes Reguladoras de Genes , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Antígenos CD28/metabolismo , Transdução de Sinais , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Ligante CD27/genética , Ligante CD27/metabolismo , Linfócitos T CD8-PositivosRESUMO
Relapse is the leading cause of death after allogeneic hematopoietic stem cell transplantation (HCT) for leukemia. T cells engineered by gene transfer to express T cell receptors (TCR; TCR-T) specific for hematopoietic-restricted minor histocompatibility (H) antigens may provide a potent selective anti-leukemic effect post-HCT. We conducted a phase I clinical trial employing a novel TCR-T product targeting the minor H antigen HA-1 to treat or consolidate treatment of persistent or recurrent leukemia and myeloid neoplasms. The primary objective was to evaluate the feasibility and safety of administration of HA-1 TCR-T post-HCT. CD8+ and CD4+ T cells expressing the HA-1 TCR and a CD8-co-receptor were successfully manufactured from HA-1 disparate HCT donors. One or more infusions of HA-1 TCR-T following lymphodepleting chemotherapy were administered to nine HCT recipients who had developed disease recurrence post-HCT. TCR-T cells expanded and persisted in vivo after adoptive transfer. No dose-limiting toxicities occurred. Although the study was not designed to assess efficacy, four patients achieved or maintained complete remissions following lymphodepletion and HA-1 TCR-T, with one ongoing at >2 years. Single-cell RNA sequencing of relapsing/progressive leukemia after TCR-T therapy identified upregulated molecules associated with T cell dysfunction or cancer cell survival. HA-1 TCR-T therapy appears feasible and safe and shows preliminary signals of efficacy. This clinical trial is registered at clinicaltrials.gov as NCT03326921.
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INTRODUCTION: Transplacental fetal cell transfer results in the engraftment of fetal-origin cells in the pregnant woman's body, a phenomenon termed fetal microchimerism. Increased fetal microchimerism measured decades postpartum is implicated in maternal inflammatory disease. Understanding which factors cause increased fetal microchimerism is therefore important. During pregnancy, circulating fetal microchimerism and placental dysfunction increase with increasing gestational age, particularly towards term. Placental dysfunction is reflected by changes in circulating placenta-associated markers, specifically placental growth factor (PlGF), decreased by several 100 pg/mL, soluble fms-like tyrosine kinase-1 (sFlt-1), increased by several 1000 pg/mL, and the sFlt-1/PlGF ratio, increased by several 10 (pg/mL)/(pg/mL). We investigated whether such alterations in placenta-associated markers correlate with an increase in circulating fetal-origin cells. MATERIAL AND METHODS: We included 118 normotensive, clinically uncomplicated pregnancies (gestational age 37+1 up to 42+2 weeks' gestation) pre-delivery. PlGF and sFlt-1 (pg/mL) were measured by Elecsys® Immunoassays. We extracted DNA from maternal and fetal samples and genotyped four human leukocyte antigen loci and 17 other autosomal loci. Paternally inherited, unique fetal alleles served as polymerase chain reaction (PCR) targets for detecting fetal-origin cells in maternal buffy coat. Fetal-origin cell prevalence was assessed by logistic regression, and quantity by negative binomial regression. Statistical exposures included gestational age (weeks), PlGF (100 pg/mL), sFlt-1 (1000 pg/mL) and the sFlt-1/PlGF ratio (10 (pg/mL)/(pg/mL)). Regression models were adjusted for clinical confounders and PCR-related competing exposures. RESULTS: Gestational age was positively correlated with fetal-origin cell quantity (DRR = 2.2, P = 0.003) and PlGF was negatively correlated with fetal-origin cell prevalence (odds ratio [OR]100 = 0.6, P = 0.003) and quantity (DRR100 = 0.7, P = 0.001). The sFlt-1 and the sFlt-1/PlGF ratios were positively correlated with fetal-origin cell prevalence (OR1000 = 1.3, P = 0.014 and OR10 = 1.2, P = 0.038, respectively), but not quantity (DRR1000 = 1.1, P = 0.600; DRR10 = 1.1, P = 0.112, respectively). CONCLUSIONS: Our results suggest that placental dysfunction as evidenced by placenta-associated marker changes, may increase fetal cell transfer. The magnitudes of change tested were based on ranges in PlGF, sFlt-1 and the sFlt-1/PlGF ratio previously demonstrated in pregnancies near and post-term, lending clinical significance to our findings. Our results were statistically significant after adjusting for confounders including gestational age, supporting our novel hypothesis that underlying placental dysfunction potentially is a driver of increased fetal microchimerism.
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Placenta , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Adulto , Fator de Crescimento Placentário , Prevalência , Biomarcadores , Terceiro Trimestre da Gravidez , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Pré-Eclâmpsia/diagnósticoRESUMO
Exposure to non-inherited maternal antigens (NIMA) during the fetal period induces lifelong split tolerance to grafts expressing these allo-antigens. In adult mice, the production of extracellular vesicles (EVs) from maternal microchimeric cells causes cross-decoration (XD) of offspring dendritic cells (DC) with NIMA and upregulation of PD-L1, contributing to NIMA tolerance. To see how this may apply to humans, we tested NIMA acquisition by fetal DCS in human cord blood. The average percentage of NIMA-XD among total DCs was 2.6% for myeloid and 4.5% for Plasmacytoid DC. These cells showed higher PD-L1 expression than their non-XD counterparts (mDC: p = .0016; pDC: p = .024). We detected CD9+ EVs bearing NIMA and PD-L1 in cord blood. To determine if this immune regulatory mechanism persists beyond the pregnancy, we analyzed NIMA-expressing kidney and liver transplant recipients. We found donor antigen XD DCs in peripheral blood and graft-infiltrating DCs. As in cord blood, the pattern of donor antigen expression was punctate, and PD-L1 expression was upregulated, likely due to both protein and miRNA acquired from EV. Our findings support a mechanism for split tolerance to NIMAs that develops during pregnancy and is recapitulated in adult transplant recipients.
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Vesículas Extracelulares , Transplante de Órgãos , Animais , Antígenos , Antígeno B7-H1 , Células Dendríticas , Feminino , Sangue Fetal , Tolerância Imunológica , Camundongos , Gravidez , Linfócitos T Reguladores , Tolerância ao TransplanteRESUMO
ABSTRACT: Biliary atresia (BA) is a rare disorder of unknown etiology. There is a debate as to whether maternal microchimerism plays a significant role in the development of BA or in graft tolerance after liver transplantation. Here, we performed quantitative-PCR-based assays for liver tissues of children with BA and other diseases. Maternal cells were detected in 4/13 and 1/3 of the BA and control groups, respectively. The estimated number of maternal cells ranged between 0 and 34.7 per 106 total cells. The frequency and severity of maternal microchimerism were similar between the BA and control groups, and between patients with and without acute rejection of maternal grafts. These results highlight the high frequency of maternal microchimerism in the liver. This study provides no evidence for roles of microchimerism in the etiology of BA or in graft tolerance. Thus, the biological consequences of maternal microchimerism need to be clarified in future studies.
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Atresia Biliar , Transplante de Fígado , Atresia Biliar/etiologia , Atresia Biliar/cirurgia , Criança , Quimerismo , Humanos , Fígado , Transplante de Fígado/efeitos adversosRESUMO
HLA class II genes provide the strongest genetic contribution to rheumatoid arthritis (RA). HLA-DRB1 alleles encoding the sequence DERAA are RA-protective. Paradoxically, RA risk is increased in women with DERAA+ children born prior to onset. We developed a sensitive qPCR assay specific for DERAA, and found 53% of DERAA-/- women with RA had microchimerism (Mc; pregnancy-derived allogeneic cells) carrying DERAA (DERAA-Mc) vs. 6% of healthy women. DERAA-Mc quantities correlated with an RA-risk genetic background including DERAA-binding HLA-DQ alleles, early RA onset, and aspects of RA severity. CD4+ T cells showed stronger response against DERAA+ vs. DERAA- allogeneic cell lines in vitro, in line with an immunogenic role of allogeneic DERAA. Results indicate a model where DERAA-Mc activates DERAA-directed T cells that are naturally present in DERAA-/- individuals and can have cross-reactivity against joint antigens. Moreover, we provide an explanation for the enigmatic observation that the same HLA sequence differentially affects RA risk through Mendelian inheritance vs. microchimeric cell acquisition.
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Artrite Reumatoide/imunologia , Antígenos HLA-DQ/imunologia , Cadeias HLA-DRB1/genética , Adulto , Alelos , Células Alógenas , Quimerismo , Reações Cruzadas , Epitopos/genética , Feminino , Predisposição Genética para Doença , Cadeias HLA-DRB1/metabolismo , Humanos , Linfócitos T/imunologiaRESUMO
OBJECTIVES: Microchimerism is the presence of a small quantity of cells or DNA from a genetically distinct individual. This phenomenon occurs with bidirectional maternal-fetal exchange during pregnancy. Microchimerism can persist for decades after delivery and have long-term health implications. However, little is known about why microchimerism is detectable at varying levels in different individuals. We examine the variability and the following potential determinants of maternal-origin microchimerism (MMc) in young women in the Philippines: gestational duration (in utero exposure to MMc), history of being breastfed (postpartum exposure to MMc), maternal telomere length (maternal cells' ability to replicate and persist), and participant's pregnancies in young adulthood (effect of adding fetal-origin microchimerism to preexisting MMc). MATERIALS AND METHODS: Data are from the Cebu Longitudinal Health and Nutrition Survey, a population-based study of infant feeding practices and long-term health outcomes. We quantified MMc using quantitative PCR (qPCR) in 89 female participants, ages 20-22, and analyzed these data using negative binomial regression. RESULTS: In a multivariate model including all predictors, being breastfed substantially predicted decreased MMc (detection rate ratio = 0.15, p = 0.007), and there was a trend of decreasing MMc in participants who had experienced more pregnancies (detection rate ratio = 0.55, p = 0.057). DISCUSSION: These results might be explained by breastfeeding having lasting impact on immune regulatory networks, thus reducing MMc persistence. MMc may also decrease in response to the introduction of fetal-origin microchimerism with pregnancies experienced in adulthood.
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Quimerismo , Gravidez/genética , Gravidez/estatística & dados numéricos , Adulto , Antropologia Física , Aleitamento Materno/estatística & dados numéricos , Estudos de Coortes , DNA/análise , DNA/classificação , DNA/genética , Feminino , Humanos , Tolerância Imunológica/genética , Troca Materno-Fetal/genética , Filipinas , Telômero/genética , Adulto JovemRESUMO
Background: A mother's infection with placental malaria (PM) can affect her child's susceptibility to malaria, although the mechanism remains unclear. The fetus acquires a small amount of maternal cells and DNA known as maternal microchimerism (MMc), and we hypothesized that PM increases MMc and that MMc alters risk of Plasmodium falciparum malaria during infancy. Methods: In a nested cohort from Muheza, Tanzania, we evaluated the presence and level of cord blood MMc in offspring of women with and without PM. A maternal-specific polymorphism was identified for each maternal-infant pair, and MMc was assayed by quantitative polymerase chain reaction. The ability of MMc to predict malaria outcomes during early childhood was evaluated in longitudinal models. Results: Inflammatory PM increased the detection rate of MMc among offspring of primigravidae and secundigravidae, and both noninflammatory and inflammatory PM increased the level of MMc. Detectable MMc predicted increased risk of positive blood smear but, interestingly, decreased risk of symptomatic malaria and malaria hospitalization. Conclusions: The acquisition of MMc may result in increased malaria infection but protection from malaria disease. Future studies should be directed at the cellular component of MMc, with attention to its ability to directly or indirectly coordinate anti-malarial immune responses in the offspring.
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Quimerismo/estatística & dados numéricos , Suscetibilidade a Doenças , Malária Falciparum/genética , Doenças Placentárias/genética , Complicações Parasitárias na Gravidez/genética , Adulto , Pré-Escolar , Estudos de Coortes , Feminino , Hospitalização , Humanos , Lactente , Recém-Nascido , Malária Falciparum/epidemiologia , Masculino , Doenças Placentárias/epidemiologia , Plasmodium falciparum , Gravidez , Complicações Parasitárias na Gravidez/epidemiologia , Prevalência , Adulto JovemAssuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sobrevivência de Enxerto , Adolescente , Adulto , Células Sanguíneas , Células da Medula Óssea , Bussulfano/análogos & derivados , Bussulfano/uso terapêutico , Criança , Ciclofosfamida/uso terapêutico , Feminino , Hematopoese , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Quimeras de Transplante , Condicionamento Pré-Transplante/métodos , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico , Irradiação Corporal Total , Adulto JovemRESUMO
Fetal microchimerism (FMc) arises when fetal cells enter maternal circulation, potentially persisting for decades. Increased FMc is associated with fetal growth restriction, preeclampsia, and anti-angiogenic shift in placenta-associated proteins in diabetic and normotensive term pregnancies. The two-stage model of preeclampsia postulates that placental dysfunction causes such shift in placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFLt-1), triggering maternal vascular inflammation and endothelial dysfunction. We investigated whether anti-angiogenic shift, fetal sex, fetal growth restriction, and severe maternal hypertension correlate with FMc in hypertensive disorders of pregnancy with new-onset features (n = 125). Maternal blood was drawn pre-delivery at > 25 weeks' gestation. FMc was detected by quantitative polymerase chain reaction targeting paternally inherited unique fetal alleles. PlGF and sFlt-1 were measured by immunoassay. We estimated odds ratios (ORs) by logistic regression and detection rate ratios (DRRs) by negative binomial regression. PlGF correlated negatively with FMc quantity (DRR = 0.2, p = 0.005) and female fetal sex correlated positively with FMc prevalence (OR = 5.0, p < 0.001) and quantity (DRR = 4.5, p < 0.001). Fetal growth restriction no longer correlated with increased FMc quantity after adjustment for correlates of placental dysfunction (DRR = 1.5, p = 0.272), whereas severe hypertension remained correlated with both FMc measures (OR = 5.5, p = 0.006; DRR = 6.3, p = 0.001). Our findings suggest that increased FMc is independently associated with both stages of the two-stage preeclampsia model. The association with female fetal sex has implications for microchimerism detection methodology. Future studies should target both male and female-origin FMc and focus on clarifying which placental mechanisms impact fetal cell transfer and how FMc impacts the maternal vasculature.
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Hipertensão , Pré-Eclâmpsia , Proteínas da Gravidez , Gravidez , Feminino , Masculino , Humanos , Fator de Crescimento Placentário/metabolismo , Retardo do Crescimento Fetal , Placenta/metabolismo , Proteínas da Gravidez/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Biomarcadores/metabolismoRESUMO
Increasing mixed chimerism (reemerging recipient cells) after allogeneic hematopoietic cell transplant (allo-HCT) can indicate relapse, the leading factor determining mortality in blood malignancies. Most clinical chimerism tests have limited sensitivity and are primarily designed to monitor engraftment. We developed a panel of quantitative polymerase chain reaction assays using TaqMan chemistry capable of quantifying chimerism in the order of 1 in a million. At such analytic sensitivity, we hypothesized that it could inform on relapse risk. As a proof-of-concept, we applied our panel to a retrospective cohort of patients with acute leukemia who underwent allo-HCT with known outcomes. Recipient cells in bone marrow aspirates (BMAs) remained detectable in 97.8% of tested samples. Absolute recipient chimerism proportions and rates at which these proportions increased in BMAs in the first 540 days after allo-HCT were associated with relapse. Detectable measurable residual disease (MRD) via flow cytometry in BMAs after allo-HCT showed limited correlation with relapse. This correlation noticeably strengthened when combined with increased recipient chimerism in BMAs, demonstrating the ability of our ultrasensitive chimerism assay to augment MRD data. Our technology reveals an underappreciated usefulness of clinical chimerism. Used side by side with MRD assays, it promises to improve identification of patients with the highest risk of disease reoccurrence for a chance of early intervention.
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Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Humanos , Transplante Homólogo , Quimerismo , Estudos Retrospectivos , RecidivaRESUMO
PROBLEM: Preeclampsia (PE) is associated with an increased risk of maternal cardiovascular disease (CVD), however, it is unclear whether this is due to shared underlying physiology or changes which occur during the disease process. Fetal microchimerism (FMc) within the maternal circulation can durably persist decades after pregnancy, is known to occur at greater frequency in PE, and can potentially affect local and systemic immune programming, thus changes in cellular FMc may provide a mechanism for long-term health outcomes associated with PE. METHOD OF STUDY: We investigated whether PE is associated with alterations in FMc immune and stem cell populations. We analyzed maternal peripheral blood mononuclear cells (PBMC) from PE cases (n = 16) and matched controls from normal pregnancies (n = 16), from which immune and stem cell subsets were isolated by flow cytometry. Genomic DNA was extracted from total PMBC and individual cell subsets, and FMc frequency was quantified by quantitative polymerase chain reaction assays targeting a fetal-specific non-shared polymorphism identified from family genotyping. RESULTS: There was a significant increase in FMc concentration in immune cell subsets in PE cases compared to controls, predominantly in B cell, and NK cell lymphocyte populations. There was no significant difference in FMc frequency or concentration within the stem cell population between PE and controls. CONCLUSIONS: The altered concentrations of immune cells within FMc in the maternal blood provides a potential mechanism for the inflammation which occurs during PE to induce long-lasting changes to the maternal immune system and may potentially promote chronic maternal disease.
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Leucócitos Mononucleares , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Quimerismo , Feto , Células-TroncoRESUMO
Bidirectional exchange of cells between mother and fetus establishes microchimerism (Mc). Mc can persist for decades and is associated with later-life health and disease. Greater fetal Mc is detected in the maternal compartment in preeclampsia (PE), but whether maternal Mc (MMC) in umbilical cord blood (CB) is altered in PE is unknown. We evaluated MMc in CB from normal and PE pregnancies. DNA from CB mononuclear cells following placental delivery (n = 36 PE, n = 37 controls) and maternal blood was extracted and genotyped. MMc, quantified by qPCR assays targeting maternal-specific nonshared polymorphisms in CB, was compared using logistic and negative binomial regression models. Clinically and statistically relevant confounders were included, and included the total number of cell equivalents tested, gravidity, mode of delivery, birthweight, and fetal sex. PE participants delivered at earlier gestational ages, with higher Cesarean rates, and lower infant birthweights. CB MMc detection was similar between PE and controls (52.8% vs. 51.3%, respectively, p = 0.90) and unchanged after adjustment for confounders. MMc concentration was not different between groups (mean 73.7 gEq/105 gEq in PE vs. mean 22.8 gEq/105 in controls, p = 0.56), including after controlling for confounders (p = 0.64). There was no difference in CB MMc detection or concentration between PE and normal pregnancies, despite previously noted greater fetal Mc in the maternal compartment. This suggests possible differential transfer of cells at the maternal fetal interface in PE. Phenotypic evaluation of Mc cells may uncover underlying mechanisms for differential cellular exchange between mother and fetus in PE.
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Placenta , Pré-Eclâmpsia , Gravidez , Humanos , Feminino , Quimerismo , Mães , Cordão Umbilical , Sangue FetalRESUMO
Fetal microchimerism (FMc) arises during pregnancy as fetal cells enter maternal circulation and remain decades postpartum. Circulating FMc is increased in preeclampsia, fetal growth restriction, and as we recently showed, is associated with biomarkers of placental dysfunction in normotensive term pregnancies. Diabetes mellitus (DM) also correlates with placental dysfunction. We hypothesize that poor glucose control and markers of placental dysfunction are associated with increased circulating FMc in diabetic pregnancies. We included 122 pregnancies preceding active labor (pregestational DM, n = 77, gestational DM (GDM), n = 45) between 2001 and 2017. Maternal and fetal samples were genotyped for various human leukocyte antigen (HLA) loci, and other polymorphisms to identify fetus-specific alleles. We used validated polymerase chain reaction (PCR) assays to quantify FMc in maternal peripheral blood buffy coat. Negative binomial regression with adjustment for confounders was used to assess FMc quantity. In pregestational DM, increased circulating FMc correlated with elevation of HbA1c (≥ 6.0 %) (detection rate ratio (DRR) = 4.9, p = 0.010) and a 1000 pg/mL rise in the anti-angiogenic biomarker soluble fms-like tyrosine kinase-1 (sFlt-1) (DRR = 1.1, p = 0.011). In GDM, increased FMc correlated with elevated 2-hour oral glucose tolerance test results (DRR = 2.3, p = 0.046) and birthweight < 10th or > 90th percentile (DRR = 4.2, p = 0.049). These findings support our novel hypothesis that FMc correlates with poor glucose control and various aspects of placental dysfunction in DM. Whether increased FMc in pregnancies with poor glucose control and placental dysfunction contributes to the risk of preeclampsia in diabetic pregnancies and to the increased risk of chronic cardiovascular disease later in life remains to be investigated.
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Diabetes Mellitus , Doenças Placentárias , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Placenta , Glicemia , Quimerismo , Feto , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , BiomarcadoresRESUMO
Fetal cells cross the placenta during pregnancy and some have the ability to persist in maternal organs and circulation long-term, a phenomenon termed fetal microchimerism. These cells often belong to stem cell or immune cell lineages. The long-term effects of fetal microchimerism are likely mixed, potentially depending on the amount of fetal cells transferred, fetal-maternal histocompatibility and fetal cell-specific properties. Both human and animal data indicate that fetal-origin cells partake in tissue repair and may benefit maternal health overall. On the other hand, these cells have been implicated in inflammatory diseases by studies showing increased fetal microchimerism in women with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. During pregnancy, preeclampsia is associated with increased cell-transfer between the mother and fetus, and an increase in immune cell subsets. In the current review, we discuss potential mechanisms of transplacental transfer, including passive leakage across the compromised diffusion barrier and active recruitment of cells residing in the placenta or fetal circulation. Within the conceptual framework of the two-stage model of preeclampsia, where syncytiotrophoblast stress is a common pathophysiological pathway to maternal and fetal clinical features of preeclampsia, we argue that microchimerism may represent a mechanistic link between stage 1 placental dysfunction and stage 2 maternal cardiovascular inflammation and endothelial dysfunction. Finally, we postulate that fetal microchimerism may contribute to the known association between placental syndromes and increased long-term maternal cardiovascular disease risk. Fetal microchimerism research represents an exciting opportunity for developing new disease biomarkers and targeted prophylaxis against maternal diseases.
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Troca Materno-Fetal , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Placenta , Quimerismo , FetoRESUMO
Tumor heterogeneity is a major driver of cancer progression. In epithelial-derived malignancies, carcinoma-associated fibroblasts (CAFs) contribute to tumor heterogeneity by depositing extracellular matrix (ECM) proteins that dynamically remodel the tumor microenvironment (TME). Ewing sarcomas (EwS) are histologically monomorphous, mesenchyme-derived tumors that are devoid of CAFs. Here we identify a previously uncharacterized subpopulation of transcriptionally distinct EwS tumor cells that deposit pro-tumorigenic ECM. Single cell analyses revealed that these CAF-like cells differ from bulk EwS cells by their upregulation of a matrisome-rich gene signature that is normally repressed by EWS::FLI1, the oncogenic fusion transcription factor that underlies EwS pathogenesis. Further, our studies showed that ECM-depositing tumor cells express the cell surface marker CD73, allowing for their isolation ex vivo and detection in situ. Spatial profiling of tumor xenografts and patient biopsies demonstrated that CD73 + EwS cells and tumor cell-derived ECM are prevalent along tumor borders and invasive fronts. Importantly, despite loss of EWS::FLI1-mediated gene repression, CD73 + EwS cells retain expression of EWS::FLI1 and the fusion-activated gene signature, as well as tumorigenic and proliferative capacities. Thus, EwS tumor cells can be reprogrammed to adopt CAF-like properties and these transcriptionally and phenotypically distinct cell subpopulations contribute to tumor heterogeneity by remodeling the TME.
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PURPOSE: Despite limited genetic and histologic heterogeneity, Ewing sarcoma (EwS) tumor cells are transcriptionally heterogeneous and display varying degrees of mesenchymal lineage specification in vitro. In this study, we investigated if and how transcriptional heterogeneity of EwS cells contributes to heterogeneity of tumor phenotypes in vivo. EXPERIMENTAL DESIGN: Single-cell proteogenomic-sequencing of EwS cell lines was performed and integrated with patient tumor transcriptomic data. Cell subpopulations were isolated by FACS for assessment of gene expression and phenotype. Digital spatial profiling and human whole transcriptome analysis interrogated transcriptomic heterogeneity in EwS xenografts. Tumor cell subpopulations and matrix protein deposition were evaluated in xenografts and patient tumors using multiplex immunofluorescence staining. RESULTS: We identified CD73 as a biomarker of highly mesenchymal EwS cell subpopulations in tumor models and patient biopsies. CD73+ tumor cells displayed distinct transcriptional and phenotypic properties, including selective upregulation of genes that are repressed by EWS::FLI1, and increased migratory potential. CD73+ cells were distinguished in vitro and in vivo by increased expression of matrisomal genes and abundant deposition of extracellular matrix (ECM) proteins. In epithelial-derived malignancies, ECM is largely deposited by cancer-associated fibroblasts (CAF), and we thus labeled CD73+ EwS cells, CAF-like tumor cells. Marked heterogeneity of CD73+ EwS cell frequency and distribution was detected in tumors in situ, and CAF-like tumor cells and associated ECM were observed in peri-necrotic regions and invasive foci. CONCLUSIONS: EwS tumor cells can adopt CAF-like properties, and these distinct cell subpopulations contribute to tumor heterogeneity by remodeling the tumor microenvironment. See related commentary by Kuo and Amatruda, p. 5002.
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Fibroblastos Associados a Câncer , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/patologia , Fibroblastos Associados a Câncer/metabolismo , Microambiente Tumoral/genética , Linhagem Celular Tumoral , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Perfilação da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Genomic chimerism represents co-existing cells with different genotypes and has diagnostic significance in transplant engraftment monitoring, residual cancer detection, and other contexts. We previously described an approach to chimerism detection by interrogating variably present or absent genomic loci using single-molecule molecular inversion probes (smMIPs) and next-generation sequencing, which provided ultrasensitive limits of detection (<1 in 10,000 cells) but was not reliably quantitative. Herein, smMIP testing was modified to accurately quantitate chimeric cells by incorporating copy number neutral control loci for data normalization and computationally modeling cell mixtures from individual-specific genotypes. Data demonstrate precision and accuracy over three orders of magnitude (0.01% to 50% chimerism). Seventy hematopoietic stem cell transplant specimens from single (n = 42) or double (n = 28) donors were evaluated, benchmarking smMIP against conventional variable number tandem repeat (VNTR) analysis and an unrelated, ultrasensitive polymorphism-specific quantitative PCR (PS-qPCR) assay. Quantitative concordance of all three assays was high (P < 0.0005, Pearson correlation coefficient), although smMIP correlated better with VNTR testing than PS-qPCR. smMIP and PS-qPCR collectively identified low-level chimerism in all specimens testing negative by VNTR (n = 41 and n = 45 of 48 specimens, respectively). This work demonstrates the feasibility of smMIP-based chimerism testing for quantitative and ultrasensitive measurement of genomic chimerism at practical levels approaching one in one million cells, and cross-validates the approach.
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Quimerismo , Transplante de Células-Tronco Hematopoéticas , Variações do Número de Cópias de DNA/genética , Genômica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
Introduction: We aimed to quantify the DNA of maternal chimeric (MC) cells in the peripheral blood of the BA patients and investigated the impact on the outcome. Methods: Patients with progressive jaundice because of no bile flow, which necessitated liver transplantation, or who showed inadequate bile flow with or without episodes of cholangitis and progressive hepatic fibrosis and portal hypertension were classified into the poor group. Those with adequate bile flow with completely normal liver function tests beyond 2 years were classified into the good group. The qPCR were separately carried out in buffy coat samples and plasma samples, targeting the non-inherited maternal HLA alleles in the DNA samples. Results: MC-DNA was present in the buffy coat (10-328 gEq per 106 host cells) in seven patients. There was no MC-DNA in the remaining five patients. MC-DNA (214-15,331 gEq per 106 host cells) was observed in the plasma of five patients. The quantity of MC-DNA in the buffy coat showed a significant difference between the two prognostic groups (p = 0.018), whereas there was no significant difference in the quantity of MC-DNA in plasma (p = 0.205). MC-DNA in the buffy coat was significantly associated with the outcome (p = 0.028), whereas MC-DNA in the plasma did not influence the outcome (p = 0.56). Conclusions: Poor outcomes in BA were correlated with circulating maternal chimeric lymphocytes.
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Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors that influence MMc detection and level across infancy and the effect of MMc on T cell responses to bacillus Calmette-Guérin (BCG) vaccination in a cohort of HIV-exposed, uninfected and HIV-unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day 1, and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy, with levels ranging from 0 to 1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal and infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc level in HIV-exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence the level of MMc, which may subsequently affect infant T cell responses.