RESUMO
Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.
Assuntos
Doença Enxerto-Hospedeiro , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/microbiologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T CD4-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Microbiota/imunologia , Seleção Clonal Mediada por Antígeno , Transplante Homólogo , Teorema de Bayes , Transplante de Células-Tronco/efeitos adversos , Camundongos Endogâmicos BALB C , Microbioma Gastrointestinal/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversosRESUMO
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.
Assuntos
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
ABSTRACT: Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognized, in which brain-infiltrating donor major histocompatibility complex (MHC) class II+ bone marrow-derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early after transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients, in which CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signaling after transplant failed to reverse GVHD-induced behavioral changes. Moreover, we observed aberrant behavior in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr-/- grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Animais , Doenças Neuroinflamatórias , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos T CD4-Positivos , Macrófagos/patologia , Receptores Proteína Tirosina Quinases , Receptores de Fator Estimulador de ColôniasRESUMO
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.
RESUMO
Adoptive transfer of T cells expressing chimeric antigen receptors (CAR-T) effectively treats refractory hematologic malignancies in a subset of patients but can be limited by poor T-cell expansion and persistence in vivo. Less differentiated T-cell states correlate with the capacity of CAR-T to proliferate and mediate antitumor responses, and interventions that limit tumor-specific T-cell differentiation during ex vivo manufacturing enhance efficacy. NOTCH signaling is involved in fate decisions across diverse cell lineages and in memory CD8+ T cells was reported to upregulate the transcription factor FOXM1, attenuate differentiation, and enhance proliferation and antitumor efficacy in vivo. Here, we used a cell-free culture system to provide an agonistic NOTCH1 signal during naïve CD4+ T-cell activation and CAR-T production and studied the effects on differentiation, transcription factor expression, cytokine production, and responses to tumor. NOTCH1 agonism efficiently induced a stem cell memory phenotype in CAR-T derived from naïve but not memory CD4+ T cells and upregulated expression of AhR and c-MAF, driving heightened production of interleukin-22, interleukin-10, and granzyme B. NOTCH1-agonized CD4+ CAR-T demonstrated enhanced antigen responsiveness and proliferated to strikingly higher frequencies in mice bearing human lymphoma xenografts. NOTCH1-agonized CD4+ CAR-T also provided superior help to cotransferred CD8+ CAR-T, driving improved expansion and curative antitumor responses in vivo at low CAR-T doses. Our data expand the mechanisms by which NOTCH can shape CD4+ T-cell behavior and demonstrate that activating NOTCH1 signaling during genetic modification ex vivo is a potential strategy for enhancing the function of T cells engineered with tumor-targeting receptors.
Assuntos
Linfoma , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Imunoterapia Adotiva , Linfócitos T CD4-Positivos , Fatores de Transcrição , Linfoma/tratamento farmacológico , Receptores de Antígenos de Linfócitos T , Receptor Notch1/genéticaRESUMO
Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.
Assuntos
Cromatina , DNA , Cromatina/genética , DNA/genética , Sequenciamento de Cromatina por Imunoprecipitação , Análise de Sequência de DNA/métodos , Epigenômica/métodosRESUMO
The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.
Assuntos
Transplante de Medula Óssea , Ciclopropanos/uso terapêutico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Receptores X de Retinoides/agonistas , Animais , Transplante de Medula Óssea/efeitos adversos , Reposicionamento de Medicamentos , Feminino , Doença Enxerto-Hospedeiro/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/patologiaRESUMO
NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).
Assuntos
Leucemia Mieloide Aguda , Criança , Adulto Jovem , Humanos , Leucemia Mieloide Aguda/genética , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Perfilação da Expressão Gênica , Proteína 2 de Ligação ao Retinoblastoma/genéticaRESUMO
Acute myeloid leukemia (AML) patients have a wide array of cytogenetic and molecular aberrations, which can influence response to therapy. Monosomy 7 is a rare subset within pediatric AML (prevalence of <2%) that is highly associated with poor outcomes. Fusions involving the anaplastic tyrosine kinase (ALK) gene were exclusively identified in 14.3% of this high-risk cohort, while absent across all other AML. Given the dismal outcomes of monosomy 7, we evaluated the use of crizotinib, an FDA-approved tyrosine kinase inhibitor, used to treat patients with ALK fusions. Our findings suggest that crizotinib may serve as a novel therapy for these patients.
Assuntos
Leucemia Mieloide Aguda , Criança , Humanos , Deleção Cromossômica , Crizotinibe/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/uso terapêuticoRESUMO
BACKGROUND AIMS: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of 'off the shelf' Treg. RESULTS: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. DISCUSSION: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile-desirable properties that support the possibility of off-the-shelf Treg therapeutics.
Assuntos
Doença Enxerto-Hospedeiro , Linfócitos T Reguladores , Transferência Adotiva , Sangue Fetal , Fatores de Transcrição Forkhead/genética , HumanosRESUMO
The clinical success of chimeric antigen receptor (CAR) T cell therapy for CD19+ B cell malignancies can be limited by acute toxicities and immunoglobulin replacement needs due to B cell aplasia from persistent CAR T cells. Life-threatening complications include cytokine release syndrome and neurologic adverse events, the exact etiologies of which are unclear. To elucidate the underlying toxicity mechanisms and test potentially safer CAR T cells, we developed a mouse model in which human CD19 (hCD19)-specific mouse CAR T cells were adoptively transferred into mice whose normal B cells express a hCD19 transgene at hemizygous levels. Compared to homozygous hCD19 transgenic mice that have â¼75% fewer circulating B cells, hemizygous mice had hCD19 frequencies and antigen density more closely simulating human B cells. Hemizygous mice given a lethal dose of hCD19 transgene-expressing lymphoma cells and treated with CAR T cells had undetectable tumor levels. Recipients experienced B cell aplasia and antigen- and dose-dependent acute toxicities mirroring patient complications. Interleukin-6 (IL-6), interferon γ (IFN-γ), and inflammatory pathway transcripts were enriched in affected tissues. As in patients, antibody-mediated neutralization of IL-6 (and IFN-γ) blunted toxicity. Apparent behavioral abnormalities associated with decreased microglial cells point to CAR-T-cell-induced neurotoxicity. This model will prove useful in testing strategies designed to improve hCD19-specific CAR T cell safety.
Assuntos
Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Animais , Feminino , Humanos , Imunoterapia Adotiva , Interferon gama/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos TransgênicosRESUMO
One of the central challenges of transplantation is the development of alloreactivity despite the use of multiagent immunoprophylaxis. Effective control of this immune suppression-resistant T-cell activation represents one of the key unmet needs in the fields of both solid-organ and hematopoietic stem cell transplant (HCT). To address this unmet need, we have used a highly translational nonhuman primate (NHP) model to interrogate the transcriptional signature of T cells during breakthrough acute graft-versus-host disease (GVHD) that occurs in the setting of clinically relevant immune suppression and compared this to the hyperacute GVHD, which develops in unprophylaxed or suboptimally prophylaxed transplant recipients. Our results demonstrate the complex character of the alloreactivity that develops during ongoing immunoprophylaxis and identify 3 key transcriptional hallmarks of breakthrough acute GVHD that are not observed in hyperacute GVHD: (1) T-cell persistence rather than proliferation, (2) evidence for highly inflammatory transcriptional programming, and (3) skewing toward a T helper (Th)/T cytotoxic (Tc)17 transcriptional program. Importantly, the gene coexpression profiles from human HCT recipients who developed GVHD while on immunosuppressive prophylactic agents recapitulated the patterns observed in NHP, and demonstrated an evolution toward a more inflammatory signature as time posttransplant progressed. These results strongly implicate the evolution of both inflammatory and interleukin 17-based immune pathogenesis in GVHD, and provide the first map of this evolving process in primates in the setting of clinically relevant immunomodulation. This map represents a novel transcriptomic resource for further systems-based efforts to study the breakthrough alloresponse that occurs posttransplant despite immunoprophylaxis and to develop evidence-based strategies for effective treatment of this disease.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Interleucina-17/imunologia , Linfócitos T Citotóxicos , Linfócitos T Auxiliares-Indutores , Doença Aguda , Aloenxertos , Animais , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/prevenção & controle , Haplorrinos , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Masculino , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
Cleavage under targets and tagmentation (CUT&Tag) is an antibody-directed in situ chromatin profiling strategy that is rapidly replacing immune precipitation-based methods, such as chromatin immunoprecipitation-sequencing. The efficiency of the method enables chromatin profiling in single cells but is limited by the numbers of cells that can be profiled. Here, we describe a combinatorial barcoding strategy for CUT&Tag that harnesses a nanowell dispenser for simple, high-resolution, high-throughput, single-cell chromatin profiling. In this single-cell combinatorial indexing CUT&Tag (sciCUT&Tag) protocol, lightly cross-linked nuclei are bound to magnetic beads and incubated with primary and secondary antibodies in bulk and then arrayed in a 96-well plate for a first round of cellular indexing by antibody-directed Tn5 tagmentation. The sample is then repooled, mixed and arrayed across 5,184 nanowells at a density of 12-24 nuclei per well for a second round of cellular indexing during PCR amplification of the sequencing-ready library. This protocol can be completed in 1.5 days by a research technician, and we illustrate the optimized protocol by profiling histone modifications associated with developmental gene repression (H3K27me3) as well as transcriptional activation (H3K4me1-2-3) in human peripheral blood mononuclear cells and use single-nucleotide polymorphisms to facilitate collision removal. We have also used sciCUT&Tag for simultaneous profiling of multiple chromatin epitopes in single cells. The reduced cost, improved resolution and scalability of sciCUT&Tag make it an attractive platform to profile chromatin features in single cells.
Assuntos
Histonas , Leucócitos Mononucleares , Humanos , Histonas/genética , Histonas/metabolismo , Leucócitos Mononucleares/metabolismo , Cromatina/genética , Processamento de Proteína Pós-Traducional , Código das Histonas , Análise de Célula Única/métodosRESUMO
Chronic antigen stimulation is thought to generate dysfunctional CD8 T cells. Here, we identify a CD8 T cell subset in the bone marrow tumor microenvironment that, despite an apparent terminally exhausted phenotype (TPHEX), expressed granzymes, perforin, and IFN-γ. Concurrent gene expression and DNA accessibility revealed that genes encoding these functional proteins correlated with BATF expression and motif accessibility. IFN-γ+ TPHEX effectively killed myeloma with comparable efficacy to transitory effectors, and disease progression correlated with numerical deficits in IFN-γ+ TPHEX. We also observed IFN-γ+ TPHEX within CD19-targeted chimeric antigen receptor T cells, which killed CD19+ leukemia cells. An IFN-γ+ TPHEX gene signature was recapitulated in TEX cells from human cancers, including myeloma and lymphoma. Here, we characterize a TEX subset in hematological malignancies that paradoxically retains function and is distinct from dysfunctional TEX found in chronic viral infections. Thus, IFN-γ+ TPHEX represent a potential target for immunotherapy of blood cancers.
Assuntos
Neoplasias Hematológicas , Mieloma Múltiplo , Humanos , Receptor Celular 2 do Vírus da Hepatite A , Mieloma Múltiplo/metabolismo , Linfócitos T CD8-Positivos , Fenótipo , Microambiente TumoralRESUMO
Calcineurin inhibitors (CNIs) constitute the backbone of modern acute graft-versus-host disease (aGVHD) prophylaxis regimens but have limited efficacy in the prevention and treatment of chronic GVHD (cGVHD). We investigated the effect of CNIs on immune tolerance after stem cell transplantation with discovery-based single-cell gene expression and T cell receptor (TCR) assays of clonal immunity in tandem with traditional protein-based approaches and preclinical modeling. While cyclosporin and tacrolimus suppressed the clonal expansion of CD8+ T cells during GVHD, alloreactive CD4+ T cell clusters were preferentially expanded. Moreover, CNIs mediated reversible dose-dependent suppression of T cell activation and all stages of donor T cell exhaustion. Critically, CNIs promoted the expansion of both polyclonal and TCR-specific alloreactive central memory CD4+ T cells (TCM) with high self-renewal capacity that mediated cGVHD following drug withdrawal. In contrast to posttransplant cyclophosphamide (PT-Cy), CSA was ineffective in eliminating IL-17A-secreting alloreactive T cell clones that play an important role in the pathogenesis of cGVHD. Collectively, we have shown that, although CNIs attenuate aGVHD, they paradoxically rescue alloantigen-specific TCM, especially within the CD4+ compartment in lymphoid and GVHD target tissues, thus predisposing patients to cGVHD. These data provide further evidence to caution against CNI-based immune suppression without concurrent approaches that eliminate alloreactive T cell clones.
Assuntos
Inibidores de Calcineurina , Doença Enxerto-Hospedeiro , Isoantígenos , Células T de Memória , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/patologia , Animais , Camundongos , Isoantígenos/imunologia , Inibidores de Calcineurina/farmacologia , Doença Crônica , Células T de Memória/imunologia , Tacrolimo/farmacologia , Linfócitos T CD4-Positivos/imunologia , Ciclosporina/farmacologia , Feminino , Linfócitos T CD8-Positivos/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-γ secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-γ in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-γ-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy.
Assuntos
Transplante de Medula Óssea , Infecções por Citomegalovirus , Imunidade Humoral , Interleucina-6 , Antivirais , Transplante de Medula Óssea/efeitos adversos , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/metabolismo , Imunoglobulina G , Interleucina-6/metabolismo , Animais , CamundongosRESUMO
Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.
RESUMO
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.
RESUMO
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.
Assuntos
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
Autologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target. Here we demonstrate high expression of TIGIT on activated CD8+ T cells in mobilized peripheral blood stem cell grafts from patients with myeloma. To guide clinical application of TIGIT inhibition, we evaluated identical anti-TIGIT antibodies that do or do not engage FcγR and demonstrated that anti-TIGIT activity is dependent on FcγR binding. We subsequently used CRBN mice to investigate the efficacy of anti-TIGIT in combination with lenalidomide maintenance after transplantation. Notably, the combination of anti-TIGIT with lenalidomide provided synergistic, CD8+ T cell-dependent, antimyeloma efficacy. Analysis of bone marrow (BM) CD8+ T cells demonstrated that combination therapy suppressed T cell exhaustion, enhanced effector function, and expanded central memory subsets. Importantly, these immune phenotypes were specific to the BM tumor microenvironment. Collectively, these data provide a logical rationale for combining TIGIT inhibition with immunomodulatory drugs to prevent myeloma progression after ASCT.