RESUMEN
Despite advancements in antifibrotic therapy, idiopathic pulmonary fibrosis (IPF) remains a medical condition with unmet needs. Single-cell RNA sequencing (scRNA-seq) has enhanced our understanding of IPF but lacks the cellular tissue context and gene expression localization that spatial transcriptomics provides. To bridge this gap, we profiled IPF and control patient lung tissue using spatial transcriptomics, integrating the data with an IPF scRNA-seq atlas. We identified three disease-associated niches with unique cellular compositions and localizations. These include a fibrotic niche, consisting of myofibroblasts and aberrant basaloid cells, located around airways and adjacent to an airway macrophage niche in the lumen, containing SPP1+ macrophages. In addition, we identified an immune niche, characterized by distinct lymphoid cell foci in fibrotic tissue, surrounded by remodeled endothelial vessels. This spatial characterization of IPF niches will facilitate the identification of drug targets that disrupt disease-driving niches and aid in the development of disease relevant in vitro models.
Asunto(s)
Fibrosis Pulmonar Idiopática , Pulmón , Transcriptoma , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/genética , Humanos , Pulmón/patología , Pulmón/metabolismo , Macrófagos/metabolismo , Análisis de la Célula Individual , Perfilación de la Expresión Génica , Miofibroblastos/metabolismo , Miofibroblastos/patologíaRESUMEN
Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.
Asunto(s)
Linfocitos T CD8-positivos , Proliferación Celular , Dinoprostona , Linfocitos Infiltrantes de Tumor , Neoplasias , Células Madre , Escape del Tumor , Animales , Femenino , Humanos , Masculino , Ratones , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Interleucina-2 , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/prevención & control , Subtipo EP2 de Receptores de Prostaglandina E/deficiencia , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/deficiencia , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismo , Escape del Tumor/inmunologíaRESUMEN
Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.
Asunto(s)
Acuaporina 4 , Autoanticuerpos , Autoantígenos , Linfocitos B , Tolerancia Inmunológica , Neuromielitis Óptica , Animales , Humanos , Ratones , Proteína AIRE , Acuaporina 4/deficiencia , Acuaporina 4/genética , Acuaporina 4/inmunología , Acuaporina 4/metabolismo , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD40/inmunología , Centro Germinal/citología , Centro Germinal/inmunología , Neuromielitis Óptica/inmunología , Neuromielitis Óptica/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Timo/citología , Timo/inmunología , Células Epiteliales Tiroideas/inmunología , Células Epiteliales Tiroideas/metabolismo , TranscriptomaRESUMEN
The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.
Asunto(s)
Bacteriófagos , Trasplante de Células Madre Hematopoyéticas , Humanos , Bacteriófagos/genética , Heces/microbiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Bacterias/genética , Bacterias/metabolismo , Ácido Butírico/metabolismoRESUMEN
Acute graft-versus-host disease (aGvHD) is a significant complication after allogeneic hematopoietic stem cell transplantation (aHSCT), but major factors determining disease severity are not well defined yet. By combining multiplexed tissue imaging and single-cell RNA sequencing on gastrointestinal biopsies from aHSCT-treated individuals with fecal microbiome analysis, we link high microbiome diversity and the abundance of short-chain fatty acid-producing bacteria to the sustenance of suppressive regulatory T cells (Tregs). Furthermore, aGvHD severity strongly associates with the clonal expansion of mainly CD8 T cells, which we find distributed over anatomically distant regions of the gut, persistent over time, and inversely correlated with the presence of suppressive Tregs. Overall, our study highlights the pathophysiological importance of expanded CD8 T cell clones in the progression of aGvHD toward more severe clinical manifestations and strongly supports the further development of microbiome interventions as GvHD treatment via repopulation of the gut Treg niche to suppress inflammation.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Microbiota , Humanos , Enfermedad Injerto contra Huésped/patología , Microbiota/genética , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Tracto Gastrointestinal/patología , Linfocitos T CD8-positivos/patologíaRESUMEN
Repetitive pathogen exposure leads to the dominant outgrowth of T cell clones with high T cell receptor (TCR) affinity to the relevant pathogen-associated antigens. However, low-affinity clones are also known to expand and form immunological memory. While these low-affinity clones contribute less immunity to the original pathogen, their role in protection against pathogens harboring immune escape mutations remains unclear. Based on identification of the TCR repertoire and functionality landscape of naive epitope-specific CD8+ T cells, we reconstructed defined repertoires that could be followed as polyclonal populations during immune responses in vivo. We found that selective clonal expansion is governed by clear TCR avidity thresholds. Simultaneously, initial recruitment of broad TCR repertoires provided a polyclonal niche from which flexible secondary responses to mutant epitopes could be recalled. Elucidating how T cell responses develop "from scratch" is informative for the development of enhanced immunotherapies and vaccines.
Asunto(s)
Linfocitos T CD8-positivos , Reinfección , Humanos , Epítopos , Receptores de Antígenos de Linfocitos T/genética , Células Clonales , Mutación/genéticaRESUMEN
OBJECTIVE: Helicobacter pylori infection is the most prevalent bacterial infection worldwide. Besides being the most important risk factor for gastric cancer development, epidemiological data show that infected individuals harbour a nearly twofold increased risk to develop colorectal cancer (CRC). However, a direct causal and functional connection between H. pylori infection and colon cancer is lacking. DESIGN: We infected two Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive analysis of H. pylori-induced changes in intestinal immune responses and epithelial signatures via flow cytometry, chip cytometry, immunohistochemistry and single cell RNA sequencing. Microbial signatures were characterised and evaluated in germ-free mice and via stool transfer experiments. RESULTS: H. pylori infection accelerated tumour development in Apc-mutant mice. We identified a unique H. pylori-driven immune alteration signature characterised by a reduction in regulatory T cells and pro-inflammatory T cells. Furthermore, in the intestinal and colonic epithelium, H. pylori induced pro-carcinogenic STAT3 signalling and a loss of goblet cells, changes that have been shown to contribute-in combination with pro-inflammatory and mucus degrading microbial signatures-to tumour development. Similar immune and epithelial alterations were found in human colon biopsies from H. pylori-infected patients. Housing of Apc-mutant mice under germ-free conditions ameliorated, and early antibiotic eradication of H. pylori infection normalised the tumour incidence to the level of uninfected controls. CONCLUSIONS: Our studies provide evidence that H. pylori infection is a strong causal promoter of colorectal carcinogenesis. Therefore, implementation of H. pylori status into preventive measures of CRC should be considered.
Asunto(s)
Neoplasias del Colon , Infecciones por Helicobacter , Helicobacter pylori , Microbiota , Neoplasias Gástricas , Humanos , Ratones , Animales , Helicobacter pylori/genética , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Ratones Endogámicos C57BL , Carcinogénesis/patología , Neoplasias Gástricas/patología , Neoplasias del Colon/patología , Moco , Mucosa Gástrica/patologíaRESUMEN
Engagement of the inhibitory T cell receptor programmed cell death protein 1 (PD-1) associates with dysfunctional states of pathogen- or tumor-specific T cells. Accordingly, systemic antibody-mediated blockade of PD-1 has become a central target for immunotherapies but is also associated with severe toxicities due to loss of peripheral tolerance. Therefore, selective ablation of PD-1 expression on adoptively transferred T cells through direct genetic knockout (KO) is currently being explored as an alternative therapeutic approach. However, since PD-1 might also be required for the regulation of physiological T cell function and differentiation, the suitability of PD-1 as an engineering target is controversial. In this study, we systematically investigated the maintenance of T cell functionality after CRISPR/Cas9-mediated PD-1 KO in vivo during and after acute and chronic antigen encounter. Under all tested conditions, PD-1 ablation preserved the persistence, differentiation, and memory formation of adoptively transferred receptor transgenic T cells. Functional PD-1 KO T cells expressing chimeric antigen receptors (CARs) targeting CD19 could be robustly detected for over 390 d in a syngeneic immunocompetent mouse model, in which constant antigen exposure was provided by continuous B cell renewal, representing the longest in vivo follow-up of CAR-T cells described to date. PD-1 KO CAR-T cells showed no evidence for malignant transformation during the entire observation period. Our data demonstrate that genetic ablation of PD-1 does not impair functionality and longevity of adoptively transferred T cells per se and therefore may be pursued more generally in engineered T cell-based immunotherapy to overcome a central immunosuppressive axis.
Asunto(s)
Receptor de Muerte Celular Programada 1 , Linfocitos T , Animales , Ratones , Receptor de Muerte Celular Programada 1/genética , Proteínas Adaptadoras Transductoras de Señales , Animales Modificados Genéticamente , Anticuerpos BloqueadoresRESUMEN
BACKGROUND & AIMS: Infection with Helicobacter pylori strongly affects global health by causing chronic gastritis, ulcer disease, and gastric cancer. Although extensive research into the strong immune response against this persistently colonizing bacterium exists, the specific role of CD8+ T cells remains elusive. METHODS: We comprehensively characterize gastric H pylori-specific CD8+ T-cell responses in mice and humans by flow cytometry, RNA-sequencing, immunohistochemistry, and ChipCytometry, applying functional analyses including T-cell depletion, H pylori eradication, and ex vivo restimulation. RESULTS: We define CD8+ T-cell populations bearing a tissue-resident memory (TRM) phenotype, which infiltrate the gastric mucosa shortly after infection and mediate pathogen control by executing antigen-specific effector properties. These induced CD8+ tissue-resident memory T cells (TRM cells) show a skewed T-cell receptor beta chain usage and are mostly specific for cytotoxin-associated gene A, the distinctive oncoprotein injected by H pylori into host cells. As the infection progresses, we observe a loss of the TRM phenotype and replacement of CD8+ by CD4+ T cells, indicating a shift in the immune response during the chronic infection phase. CONCLUSIONS: Our results point toward a hitherto unknown role of CD8+ T-cell response in this bacterial infection, which may have important clinical implications for treatment and vaccination strategies against H pylori.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Humanos , Animales , Ratones , Linfocitos T CD8-positivos , Linfocitos T CD4-Positivos , Estómago , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/microbiología , Antígenos Bacterianos , Proteínas BacterianasRESUMEN
The development of ultra-thin flat liquid sheets capable of running in vacuum has provided an exciting new target for X-ray absorption spectroscopy in the liquid and solution phases. Several methods have become available for delivering in-vacuum sheet jets using different nozzle designs. We compare the sheets produced by two different types of nozzle; a commercially available borosillicate glass chip using microfluidic channels to deliver colliding jets, and an in-house fabricated fan spray nozzle which compresses the liquid on an axis out of a slit to achieve collision conditions. We find in our tests that both nozzles are suitable for use in X-ray absorption spectroscopy with the fan spray nozzle producing thicker but more stable jets than the commercial nozzle. We also provide practical details of how to run these nozzles in vacuum.
RESUMEN
The importance of T cells in controlling SARS-CoV-2 infections has been demonstrated widely, but insights into the quality of these responses are still limited due to technical challenges. Indeed, understanding the functionality of the T-cell receptor (TCR) repertoire of a polyclonal antigen-specific population still requires the tedious work of T-cell cloning or TCR re-expression and subsequent characterization. In this work, we show that it is possible to discriminate highly functional and bystander TCRs based on gene signatures of T-cell activation induced by recent peptide stimulation. SARS-CoV-2-specific TCRs previously identified by cytokine release after peptide restimulation and subsequent single-cell RNA sequencing were re-expressed via CRISPR-Cas9-mediated gene editing into a Jurkat-based reporter cell line system suitable for high-throughput screening. We could observe differences in SARS-CoV-2 epitope recognition as well as a wide range of functional avidities. By correlating these in vitro TCR engineered functional data with the transcriptomic profiles of the corresponding TCR-expressing parental T cells, we could validate that gene signatures of recent T-cell activation accurately identify and predict truly SARS-CoV-2-specific TCRs. In summary, this work paves the way for alternative approaches useful for the functional analysis of global antigen-specific TCR repertoires with largely improved throughput.
RESUMEN
We experimentally study the interaction between intense infrared few-cycle laser pulses and an ultrathin (â¼2 µm) flat liquid sheet of isopropanol running in vacuum. We observe a rapid decline in transmission above a critical peak intensity of 50 TW/cm2 of the initially transparent liquid sheet, and the emission of a plume of material. We find both events are due to the creation of a surface plasma and are similar to processes observed in dielectric solids. After calculating the electron density for different laser peak intensities, we find an electron scattering rate of 0.3 fs-1 in liquid isopropanol to be consistent with our data. We study the dynamics of the plasma plume to find the expansion velocity of the plume front.
RESUMEN
CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.
Asunto(s)
Linfocitos T CD8-positivos , Receptor de Muerte Celular Programada 1 , Proteínas Proto-Oncogénicas c-myb , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Autorrenovación de las Células , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Inmunoterapia , Selectina L/metabolismo , Células Precursoras de Linfocitos T/citología , Células Precursoras de Linfocitos T/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Virus/inmunologíaRESUMEN
T cell receptor (TCR) avidity is assumed to be a major determinant of the spatiotemporal fate and protective capacity of tumor-specific T cells. However, monitoring polyclonal T cell responses with known TCR avidities in vivo over space and time remains challenging. Here, we investigated the fate and functionality of tumor neoantigen-specific T cells with TCRs of distinct avidities in a well-established, reductionist preclinical tumor model and human patients with melanoma. To this end, we used polyclonal T cell transfers with in-depth characterized TCRs together with flow cytometric phenotyping in mice inoculated with MC38 OVA tumors. Transfer of T cells from retrogenic mice harboring TCRs with high avidity resulted in best tumor protection. Unexpectedly, we found that both high- and low-avidity T cells are similarly abundant within the tumor and adopt concordant phenotypic signs of exhaustion. Outside the tumor, high-avidity TCR T cells were not generally overrepresented but, instead, selectively enriched in T cell populations with intermediate PD-1 protein expression. Single-cell sequencing of neoantigen-specific T cells from two patients with melanoma-combined with transgenic reexpression of identified TCRs by CRISPR-Cas9-mediated orthotopic TCR replacement-revealed high-functionality TCRs to be enriched in T cells with RNA signatures of recent activation. Furthermore, of 130 surface protein candidates, PD-1 surface expression was most consistently enriched in functional TCRs. Together, our findings show that tumor-reactive TCRs with high protective capacity circulating in peripheral blood are characterized by a signature of recent activation.
Asunto(s)
Melanoma , Receptores de Antígenos de Linfocitos T , Animales , Antígenos de Neoplasias , Humanos , Ratones , Linfocitos T/metabolismoRESUMEN
In this protocol, we describe the use of ChipCytometry to combine RNA in situ hybridization and antibody staining for multiplexed tissue imaging of human formalin-fixed and paraffin-embedded tissue samples. The advantages of ChipCytometry are long-term storage for re-interrogation and advanced image quality by high dynamic range imaging of staining and background. A titrated pretreatment of tissue samples bypasses challenges because of the retrieval of antigens on coverslips and achieves an optimal staining quality at the minimal expense of tissue integrity. For complete details on the use and execution of this protocol, please refer to Jarosch et al. (2021).
Asunto(s)
Formaldehído , ARN , Humanos , Hibridación in Situ , ARN/genética , ARN Mensajero/genéticaRESUMEN
The avidity of TCRs for peptide-major histocompatibility complexes (pMHCs) is a governing factor in how T cells respond to antigen. TCR avidity is generally linked to T-cell functionality and there is growing evidence for distinct roles of low and high avidity T cells in different phases of immune responses. While physiological immune responses and many therapeutic T-cell products targeting infections or cancers consist of polyclonal T-cell populations with a wide range of individual avidities, the role of T-cell avidity is usually investigated only in monoclonal experimental settings. In this report, we induced polyclonal T-cell responses with a wide range of avidities toward a model epitope by altered peptide ligands, and benchmarked global avidity of physiological polyclonal populations by investigation of TCR-pMHC koff -rates. We then investigated how varying sizes and avidities of monoclonal subpopulations translate into global koff -rates. Global koff -rates integrate subclonal avidities in a predictably weighted manner and robustly correlate with the functionality of murine polyclonal T-cell populations in vitro and in vivo. Surveying the full avidity spectrum is essential to accurately assess polyclonal immune responses and inform the design of polyclonal T-cell therapeutics.
Asunto(s)
Receptores de Antígenos de Linfocitos T , Linfocitos T , Animales , Antígenos , Complejo Mayor de Histocompatibilidad , Ratones , Péptidos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8+ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8+ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8+ TCRs-classic features of protective immunity-are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8+ T cell immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , SARS-CoV-2/inmunología , Adulto , Células Cultivadas , Reacciones Cruzadas/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Epítopos Inmunodominantes/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Masculino , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.
Asunto(s)
Edición Génica , Genes Codificadores de los Receptores de Linfocitos T , Inmunoterapia , Linfocitos T/inmunología , Animales , Línea Celular , Membrana Celular/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos NOD , Transcripción GenéticaRESUMEN
Upon viral infection, natural killer (NK) cells expressing certain germline-encoded receptors are selected, expanded, and maintained in an adaptive-like manner. Currently, these are thought to differentiate along a common pathway. However, by fate mapping of single NK cells upon murine cytomegalovirus (MCMV) infection, we identified two distinct NK cell lineages that contributed to adaptive-like responses. One was equivalent to conventional NK (cNK) cells while the other was transcriptionally similar to type 1 innate lymphoid cells (ILC1s). ILC1-like NK cells showed splenic residency and strong cytokine production but also recognized and killed MCMV-infected cells, guided by activating receptor Ly49H. Moreover, they induced clustering of conventional type 1 dendritic cells and facilitated antigen-specific T cell priming early during MCMV infection, which depended on Ly49H and the NK cell-intrinsic expression of transcription factor Batf3. Thereby, ILC1-like NK cells bridge innate and adaptive viral recognition and unite critical features of cNK cells and ILC1s.
Asunto(s)
Inmunidad Adaptativa/inmunología , Linaje de la Célula/inmunología , Infecciones por Herpesviridae/inmunología , Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , MuromegalovirusRESUMEN
The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.