RESUMEN
Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14LowCD16High monocytes, accumulation of HLA-DRLow classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation.
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Infecciones por Coronavirus , Coronavirus , Pandemias , Neumonía Viral , Betacoronavirus , COVID-19 , Citometría de Flujo , Humanos , Complejo de Antígeno L1 de Leucocito , Monocitos , Células Mieloides , Estudios Prospectivos , SARS-CoV-2RESUMEN
Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1+CD274(PD-L1)+IDO1+ macrophages, which accumulated in the tumor periphery in a T cell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory T cell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states.
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Células Dendríticas/inmunología , Expresión Génica/inmunología , Monocitos/inmunología , Transcriptoma/genética , Macrófagos Asociados a Tumores/inmunología , Artritis Reumatoide/inmunología , COVID-19/inmunología , Expresión Génica/genética , Perfilación de la Expresión Génica , Humanos , Interferón gamma/inmunología , L-Aminoácido Oxidasa/metabolismo , Cirrosis Hepática/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , ARN Citoplasmático Pequeño/genética , Análisis de la Célula Individual , Linfocitos T Reguladores/inmunología , Transcriptoma/inmunologíaRESUMEN
Background: The Mixed Lymphocyte Reaction (MLR) consists in the allogeneic co-culture of monocytes derived dendritic cells (MoDCs) with T cells from another donor. This in vitro assay is largely used for the assessment of immunotherapy compounds. Nevertheless, the phenotypic changes associated with lymphocyte responsiveness under MLR have never been thoroughly evaluated. Methods: Here, we used multiplex cytokine and chemokine assays, multiparametric flow cytometry and single cell RNA sequencing to deeply characterize T cells activation and function in the context of CD4+- and CD8+-specific MLR kinetics. Results: We showed that CD4+ and CD8+ T cells in MLR share common classical markers of response such as polyfunctionality, increased proliferation and CD25 expression but differ in their kinetics and amplitude of activation as well as their patterns of cytokines secretion and immune checkpoints expression. The analysis of immunoreactive Ki-67+CD25+ T cells identified PBK, LRR1 and MYO1G as new potential markers of MLR response. Using cell-cell communication network inference and pathway analysis on single cell RNA sequencing data, we also highlighted key components of the immunological synapse occurring between T cells and the stimulatory MoDCs together with downstream signaling pathways involved in CD4+ and CD8+ T cells activation. Conclusion: These results provide a deep understanding of the kinetics of the MLR assay for CD4+ or CD8+ T cells and may allow to better characterize compounds impacting MLR and eventually identify new strategies for immunotherapy in cancer.
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Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Prueba de Cultivo Mixto de Linfocitos , Citometría de Flujo , Análisis de Secuencia de ARNRESUMEN
Aside from PD-L1 expression, biomarkers of response to immune checkpoint inhibitors (ICIs) in non-small-cell lung cancer (NSCLC) are needed. In a previous retrospective analysis, we documented that fecal Akkermansia muciniphila (Akk) was associated with clinical benefit of ICI in patients with NSCLC or kidney cancer. In the current study, we performed shotgun-metagenomics-based microbiome profiling in a large cohort of patients with advanced NSCLC (n = 338) treated with first- or second-line ICIs to prospectively validate the predictive value of fecal Akk. Baseline stool Akk was associated with increased objective response rates and overall survival in multivariate analyses, independent of PD-L1 expression, antibiotics, and performance status. Intestinal Akk was accompanied by a richer commensalism, including Eubacterium hallii and Bifidobacterium adolescentis, and a more inflamed tumor microenvironment in a subset of patients. However, antibiotic use (20% of cases) coincided with a relative dominance of Akk above 4.8% accompanied with the genus Clostridium, both associated with resistance to ICI. Our study shows significant differences in relative abundance of Akk that may represent potential biomarkers to refine patient stratification in future studies.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Akkermansia , Antígeno B7-H1 , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Receptor de Muerte Celular Programada 1 , Estudios Retrospectivos , Microambiente TumoralRESUMEN
Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale. SIGNIFICANCE: In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli-specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221.
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Neoplasias de la Vejiga Urinaria , Antígeno B7-H1 , Quimiocina CXCL13 , Escherichia coli , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoglobulina G , Músculos , Terapia Neoadyuvante , Receptor de Muerte Celular Programada 1 , Linfocitos T Colaboradores-Inductores , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Vaccination against coronavirus disease 2019 (COVID-19) relies on the in-depth understanding of protective immune responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We characterized the polarity and specificity of memory T cells directed against SARS-CoV-2 viral lysates and peptides to determine correlates with spontaneous, virus-elicited, or vaccine-induced protection against COVID-19 in disease-free and cancer-bearing individuals. A disbalance between type 1 and 2 cytokine release was associated with high susceptibility to COVID-19. Individuals susceptible to infection exhibited a specific deficit in the T helper 1/T cytotoxic 1 (Th1/Tc1) peptide repertoire affecting the receptor binding domain of the spike protein (S1-RBD), a hotspot of viral mutations. Current vaccines triggered Th1/Tc1 responses in only a fraction of all subject categories, more effectively against the original sequence of S1-RBD than that from viral variants. We speculate that the next generation of vaccines should elicit Th1/Tc1 T-cell responses against the S1-RBD domain of emerging viral variants. SIGNIFICANCE: This study prospectively analyzed virus-specific T-cell correlates of protection against COVID-19 in healthy and cancer-bearing individuals. A disbalance between Th1/Th2 recall responses conferred susceptibility to COVID-19 in both populations, coinciding with selective defects in Th1 recognition of the receptor binding domain of spike. See related commentary by McGary and Vardhana, p. 892. This article is highlighted in the In This Issue feature, p. 873.
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Factores de Restricción Antivirales , COVID-19 , Neoplasias , Linfocitos T , Anticuerpos Neutralizantes , Factores de Restricción Antivirales/inmunología , COVID-19/inmunología , Humanos , Neoplasias/complicaciones , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Linfocitos T/inmunologíaRESUMEN
Cancer patients are particularly susceptible to the development of severe Covid-19, prompting us to investigate the serum metabolome of 204 cancer patients enrolled in the ONCOVID trial. We previously described that the immunosuppressive tryptophan/kynurenine metabolite anthranilic acid correlates with poor prognosis in non-cancer patients. In cancer patients, we observed an elevation of anthranilic acid at baseline (without Covid-19 diagnosis) and no further increase with mild or severe Covid-19. We found that, in cancer patients, Covid-19 severity was associated with the depletion of two bacterial metabolites, indole-3-proprionate and 3-phenylproprionate, that both positively correlated with the levels of several inflammatory cytokines. Most importantly, we observed that the levels of acetylated polyamines (in particular N1-acetylspermidine, N1,N8-diacetylspermidine and N1,N12-diacetylspermine), alone or in aggregate, were elevated in severe Covid-19 cancer patients requiring hospitalization as compared to uninfected cancer patients or cancer patients with mild Covid-19. N1-acetylspermidine and N1,N8-diacetylspermidine were also increased in patients exhibiting prolonged viral shedding (>40 days). An abundant literature indicates that such acetylated polyamines increase in the serum from patients with cancer, cardiovascular disease or neurodegeneration, associated with poor prognosis. Our present work supports the contention that acetylated polyamines are associated with severe Covid-19, both in the general population and in patients with malignant disease. Severe Covid-19 is characterized by a specific metabolomic signature suggestive of the overactivation of spermine/spermidine N1-acetyl transferase-1 (SAT1), which catalyzes the first step of polyamine catabolism.
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COVID-19/sangre , COVID-19/patología , Neoplasias/sangre , Neoplasias/virología , Poliaminas/sangre , Acetilación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/microbiología , COVID-19/virología , Estudios de Cohortes , Citocinas/sangre , Femenino , Humanos , Mediadores de Inflamación/sangre , Masculino , Metaboloma , Persona de Mediana Edad , Propionatos/sangre , Índice de Severidad de la Enfermedad , Adulto Joven , ortoaminobenzoatos/sangreRESUMEN
The circulating metabolome provides a snapshot of the physiological state of the organism responding to pathogenic challenges. Here we report alterations in the plasma metabolome reflecting the clinical presentation of COVID-19 patients with mild (ambulatory) diseases, moderate disease (radiologically confirmed pneumonitis, hospitalization and oxygen therapy), and critical disease (in intensive care). This analysis revealed major disease- and stage-associated shifts in the metabolome, meaning that at least 77 metabolites including amino acids, lipids, polyamines and sugars, as well as their derivatives, were altered in critical COVID-19 patient's plasma as compared to mild COVID-19 patients. Among a uniformly moderate cohort of patients who received tocilizumab, only 10 metabolites were different among individuals with a favorable evolution as compared to those who required transfer into the intensive care unit. The elevation of one single metabolite, anthranilic acid, had a poor prognostic value, correlating with the maintenance of high interleukin-10 and -18 levels. Given that products of the kynurenine pathway including anthranilic acid have immunosuppressive properties, we speculate on the therapeutic utility to inhibit the rate-limiting enzymes of this pathway including indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase.
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COVID-19/sangre , Metaboloma , SARS-CoV-2/metabolismo , Anticuerpos Monoclonales Humanizados/administración & dosificación , Biomarcadores/sangre , COVID-19/diagnóstico , Femenino , Humanos , Masculino , Metabolómica , Pronóstico , Tratamiento Farmacológico de COVID-19RESUMEN
Patients with cancer are at higher risk of severe coronavirus infectious disease 2019 (COVID-19), but the mechanisms underlying virus-host interactions during cancer therapies remain elusive. When comparing nasopharyngeal swabs from cancer and noncancer patients for RT-qPCR cycle thresholds measuring acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in 1063 patients (58% with cancer), we found that malignant disease favors the magnitude and duration of viral RNA shedding concomitant with prolonged serum elevations of type 1 IFN that anticorrelated with anti-RBD IgG antibodies. Cancer patients with a prolonged SARS-CoV-2 RNA detection exhibited the typical immunopathology of severe COVID-19 at the early phase of infection including circulation of immature neutrophils, depletion of nonconventional monocytes, and a general lymphopenia that, however, was accompanied by a rise in plasmablasts, activated follicular T-helper cells, and non-naive Granzyme B+FasL+, EomeshighTCF-1high, PD-1+CD8+ Tc1 cells. Virus-induced lymphopenia worsened cancer-associated lymphocyte loss, and low lymphocyte counts correlated with chronic SARS-CoV-2 RNA shedding, COVID-19 severity, and a higher risk of cancer-related death in the first and second surge of the pandemic. Lymphocyte loss correlated with significant changes in metabolites from the polyamine and biliary salt pathways as well as increased blood DNA from Enterobacteriaceae and Micrococcaceae gut family members in long-term viral carriers. We surmise that cancer therapies may exacerbate the paradoxical association between lymphopenia and COVID-19-related immunopathology, and that the prevention of COVID-19-induced lymphocyte loss may reduce cancer-associated death.
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COVID-19/complicaciones , COVID-19/virología , Linfopenia/complicaciones , Neoplasias/complicaciones , ARN Viral/análisis , SARS-CoV-2/genética , Esparcimiento de Virus , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , ADN Bacteriano/sangre , Enterobacteriaceae/genética , Femenino , Humanos , Interferón Tipo I/sangre , Linfopenia/virología , Masculino , Micrococcaceae/genética , Persona de Mediana Edad , Nasofaringe/virología , Neoplasias/diagnóstico , Neoplasias/mortalidad , Pandemias , Pronóstico , Factores de Tiempo , Adulto JovenRESUMEN
Decision making in immuno-oncology is pivotal to adapt therapy to the tumor microenvironment (TME) of the patient among the numerous options of monoclonal antibodies or small molecules. Predicting the best combinatorial regimen remains an unmet medical need. Here, we report a multiplex functional and dynamic immuno-assay based on the capacity of the TME to respond to ex vivo stimulation with twelve immunomodulators including immune checkpoint inhibitors (ICI) in 43 human primary tumors. This "in sitro" (in situ/in vitro) assay has the potential to predict unresponsiveness to anti-PD-1 mAbs, and to detect the most appropriate and personalized combinatorial regimen. Prospective clinical trials are awaited to validate this in sitro assay.
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Inmunoterapia , Neoplasias , Humanos , Oncología Médica , Neoplasias/terapia , Estudios Prospectivos , Microambiente TumoralRESUMEN
Over the past 16 years, three coronaviruses (CoVs), severe acute respiratory syndrome CoV (SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012 and 2015, and SARS-CoV-2 in 2020, have been causing severe and fatal human epidemics. The unpredictability of coronavirus disease-19 (COVID-19) poses a major burden on health care and economic systems across the world. This is caused by the paucity of in-depth knowledge of the risk factors for severe COVID-19, insufficient diagnostic tools for the detection of SARS-CoV-2, as well as the absence of specific and effective drug treatments. While protective humoral and cellular immune responses are usually mounted against these betacoronaviruses, immune responses to SARS-CoV2 sometimes derail towards inflammatory tissue damage, leading to rapid admissions to intensive care units. The lack of knowledge on mechanisms that tilt the balance between these two opposite outcomes poses major threats to many ongoing clinical trials dealing with immunostimulatory or immunoregulatory therapeutics. This review will discuss innate and cognate immune responses underlying protective or deleterious immune reactions against these pathogenic coronaviruses.
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COVID-19/inmunología , Interacciones Microbiota-Huesped/inmunología , SARS-CoV-2/inmunología , COVID-19/diagnóstico , COVID-19/virología , Humanos , Inmunidad Celular , Inmunidad Humoral , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Factores Protectores , Factores de Riesgo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Índice de Severidad de la EnfermedadRESUMEN
Development of therapeutic antibodies in oncology has attracted much interest in the past decades. More than 30 of them have been approved and are being used to treat patients suffering from cancer. Despite encouraging results, and albeit most clinical trials aiming at evaluating monoclonal antibodies directed against TRAIL agonist receptors have been discontinued, DR4 or DR5 remain interesting targets, since these receptors are overexpressed by tumour cells and are able to trigger their death. In an effort to develop novel and specific anti-DR4 and anti-DR5 antibodies with improved properties, we used genetic immunization to express native proteins in vivo. Injection of DR4 and DR5 cDNA into the tail veins of mice elicited significant humoral anti-DR4 and anti-DR5 responses and fusions of the corresponding spleens resulted in numerous hybridomas secreting antibodies that could specifically recognize DR4 or DR5 in their native forms. All antibodies bound specifically to their targets with a very high affinity, from picomolar to nanomolar range. Among the 21 anti-DR4 and anti-DR5 monoclonal antibodies that we have produced and purified, two displayed proapoptotic properties alone, five induced apoptosis after cross-linking, four were found to potentiate TRAIL-induced apoptosis and three displayed antiapoptotic potential. The most potent anti-DR4 antibody, C#16, was assessed in vivo and was found, alone, to inhibit tumour growth in animal models. This is the first demonstration that DNA-based immunization method can be used to generate novel monoclonal antibodies targeting receptors of the TNF superfamily that may constitute new therapeutic agents.
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Anticuerpos Monoclonales/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Trasplante HeterólogoRESUMEN
Developing therapeutics that induce apoptosis in cancer cells has become an increasingly attractive approach for the past 30 years. The discovery of tumor necrosis factor (TNF) superfamily members and more specifically TNF-related apoptosis-inducing ligand (TRAIL), the only cytokine of the family capable of eradicating selectively cancer cells, led to the development of numerous TRAIL derivatives targeting death receptor 4 (DR4) and death receptor 5 (DR5) for cancer therapy. With a few exceptions, preliminary attempts to use recombinant TRAIL, agonistic antibodies, or derivatives to target TRAIL agonist receptors in the clinic have been fairly disappointing. Nonetheless, a tremendous effort, worldwide, is being put into the development of novel strategic options to target TRAIL receptors. Antibodies and derivatives allow for the design of novel and efficient agonists. We summarize and discuss here the advantages and drawbacks of the soar of TRAIL therapeutics, from the first developments to the next generation of agonistic products, with a particular insight on new concepts.