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BACKGROUND & AIMS: In most autoimmune disorders, crosstalk of B cells and CD4 T cells results in the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-soluble liver antigen (SLA) autoantibodies is associated with reduced overall survival, but the associated autoreactive CD4 T cells have not yet been characterised. Herein, we isolated and deeply characterised SLA-specific CD4 T cells in patients with AIH. METHODS: We used brief ex vivo restimulation with overlapping SLA peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNA-seq) to characterise their transcriptome and T-cell receptor (TCR) repertoire. Autoreactive TCRs were cloned and used to identify dominant SLA-derived epitopes. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing to identify their phenotypic niche. We further characterised disease-associated peripheral blood T cells by high-content flow cytometry in 42 patients with AIH and 17 controls with non-alcoholic steatohepatitis. RESULTS: Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1+CXCR5-CCR6-CD27+ phenotype. ScRNA-seq revealed their pro-inflammatory/B-helper profile. SLA81-100 and SLA177-204 contain dominant T-cell epitopes. Autoreactive TCR clonotypes were predominantly found in the memory PD-1+CXCR5-CD4 T cells, which were significantly increased in the blood of patients with AIH and supported B-cell differentiation through IL-21. Finally, we identified specific T-cell phenotypes linked to disease activity and IgG level during AIH. CONCLUSIONS: We provide a deep characterisation of rare circulating autoreactive CD4 T cells and identify their peripheral reservoir in AIH. We also propose a specific phenotype of autoreactive T cells related to AIH disease activity, which will be essential to track, delineate, and potentially target these pathogenic cells. LAY SUMMARY: One principal characteristic of autoimmune hepatitis (AIH), like for many other autoimmune diseases, is the accumulation of autoantibodies produced by B lymphocytes following their interaction with autoreactive CD4 T lymphocytes. In this study, we identified and characterised with high resolution these CD4 T cells. This will be essential to track, delineate, and potentially target them during AIH.
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Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Hepatitis Autoinmune , Adulto , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Epítopos de Linfocito T/análisis , Femenino , Hepatitis Autoinmune/sangre , Hepatitis Autoinmune/inmunología , Hepatitis Autoinmune/patología , Humanos , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores CXCR5/genética , Análisis de Secuencia de ARN , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
The recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpoint inhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further.
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Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos de Neoplasias/inmunología , Carcinoma de Células de Merkel/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Cutáneas/inmunología , Linfocitos T Citotóxicos/inmunología , Carcinoma de Células de Merkel/metabolismo , Carcinoma de Células de Merkel/patología , Células Cultivadas , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND AIMS: To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. METHODS: We used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP-specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. RESULTS: After these steps of selection the clone NAT-DP4(TK), specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4(TK) cells were frozen after 50 PDs. Thawed NAT-DP4(TK) cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. CONCLUSION: The possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations.
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Células Clonales/citología , Técnicas Citológicas/métodos , Cadenas beta de HLA-DP , Linfocitos T/citología , Animales , Donantes de Sangre , Genes Transgénicos Suicidas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Cadenas beta de HLA-DP/genética , Cadenas beta de HLA-DP/inmunología , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Ratones , Linfocitos T/inmunología , Timidina Quinasa/genética , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Antígenos CD19/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Recurrencia Local de Neoplasia/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Receptor ErbB-2/metabolismo , Adolescente , Adulto , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Terapia Molecular Dirigida , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Pronóstico , Adulto JovenRESUMEN
FcγRIIIA/CD16A, the low-affinity receptor for the IgG Fc portion expressed on human CD56(dim) NK cells and involved in Ab-dependent cell cytotoxicity, is shed upon NK cell activation. We found that recombinant a disintegrin and metalloprotease (ADAM) 17 cleaved the ectodomain of FcγRIIIA/CD16A and a peptide for which the sequence encompasses aa 191-201 of the FcγRIIIA/CD16A stalk region but not ADAM10. MALDI-TOF analysis revealed that the peptide was cleaved between Ala(195) and Val(196) (i.e., 1 aa upstream of the expected position). This location of the cleavage site was confirmed by the finding that ADAM17 failed to cleave a peptide in which Ala and Val were reversed. ADAM17 was found to be expressed on NK cells, and stimulation with PMA or N-ethyl-maleimide resulted in the shedding of FcγRIIIA/CD16A and CD62L, a specific substrate of ADAM17. Selective inhibition of ADAM17 prevented the shedding of both molecules. Moreover, the shedding of FcγRIIIA/CD16A was strongly correlated with degranulation when a wide range of CD56(dim) NK cell activating receptors were stimulated, whereas both ADAM17-dependent shedding and internalization were involved in FcγRIIIA/CD16A downmodulation when the latter was engaged. Finally, the shedding of FcγRIIIA/CD16A was restricted to activated cells, suggesting that ADAM17 acts mainly, if not exclusively, in cis. Taken together, our results demonstrated for the first time, to our knowledge, at the molecular level that ADAM17 cleaves the stalk region of FcγRIIIA/CD16A and identified its cleavage site. The shedding of FcγRIIIA/CD16A was at least partially ADAM17 dependent, and it may be considered as a marker of FcγRIIIA/CD16A-independent NK cell activation highly correlated with degranulation.
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Proteínas ADAM/metabolismo , Células Asesinas Naturales/metabolismo , Receptores de IgG/metabolismo , Proteína ADAM17 , Sitios de Unión , Células Cultivadas , Humanos , Péptidos/metabolismoRESUMEN
INTRODUCTION: Trastuzumab has been used in the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer, but its efficacy is limited by de novo or acquired resistance. Although many mechanisms have been proposed to explain resistance to trastuzumab, little is known concerning the role of the tumor microenvironment. Given the importance of antibody-dependent cellular cytotoxicity (ADCC) in the antitumor effect of trastuzumab and the abundance of adipose tissue in the breast, we investigated the impact of adipocytes on ADCC. METHODS: We set up a coculture system to study the effect of adipocytes on ADCC in vitro. The results were validated in vivo in a mouse xenograft model. RESULTS: We found that adipocytes, as well as preadipocytes, inhibited trastuzumab-mediated ADCC in HER2-expressing breast cancer cells via the secretion of soluble factors. The inhibition of ADCC was not due to titration or degradation of the antibody. We found that adipose cells decreased the secretion of interferon-γ by natural killer cells, but did not alter natural killer cells' cytotoxicity. Preincubation of breast cancer cells with the conditioned medium derived from adipocytes reduced the sensitivity of cancer cells to ADCC. Using a transcriptomic approach, we found that cancer cells undergo major modifications when exposed to adipocyte-conditioned medium. Importantly, breast tumors grafted next to lipomas displayed resistance to trastuzumab in mouse xenograft models. CONCLUSIONS: Collectively, our findings underline the importance of adipose tissue in the resistance to trastuzumab and suggest that approaches targeting the adipocyte-cancer cell crosstalk may help sensitize cancer cells to trastuzumab-based therapy.
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Adipocitos/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Trastuzumab/inmunología , Trastuzumab/farmacología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Diferenciación Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Medios de Cultivo Condicionados , Femenino , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Neoplasias Mamarias Animales , Ratones , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In France, hospital cell therapy units have not been authorised to routinely produce chimeric antigen receptor T lymphocytes (CAR-T cells), which would then be referred to as academic CAR-T cells. CAR-T cells are classified as advanced therapy medicinal products and correspond to genetically modified T lymphocytes ex vivo. The CAR-T cell production process is complex and requires scientific and technical expertise to meet the acceptance criteria of the pharmaceutical quality system. The most commonly used method for genetically modifying T lymphocytes is viral transduction (lentiviral or retroviral), which requires prior access to a batch of good manufacturing practice (GMP) grade viral vector. Because of its cost, this reagent is the main limiting factor for developing CAR-T cells. A CAR-T cell produced by an industrial company is expensive (around 350,000 per injection) and the time taken by the manufacturer to make it available to the clinician can vary from three to five weeks. By meeting the economic and ecological challenges, can academic structures improve access to CAR-T cells? In this article, we present the elements necessary for the feasibility of setting up CAR-T cell production in an academic structure.
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Inmunoterapia Adoptiva , Linfocitos T , Humanos , Inmunoterapia Adoptiva/métodos , Francia , Vectores Genéticos , Tratamiento Basado en Trasplante de Células y TejidosRESUMEN
BACKGROUND: Therapeutic options for CD19+ relapses after anti-CD19 CAR-T cells are still debated; second infusion of anti-CD19 CAR-T cells, therapeutic antibodies, or targeted therapies can be discussed. Here, we explore the immunophenotyping and lysis sensitivity of CD19+ ALL relapse after anti-CD19 CAR-T cells and propose different therapeutic options for such a high-risk disease. METHODS: Cells from successive B-ALL relapses from one patient were collected. A broad immunophenotype analysis was performed. 51Cr cytotoxic assays, and long-term killing assays were conducted using T-cell effectors that are capable of cytotoxicity through three recognition pathways: antibody-dependent cell-mediated cytotoxicity (ADCC), anti-CD19 CAR-T, and TCR. RESULTS: Previously targeted antigen expression, even if maintained, decreased in relapses, and new targetable antigens appeared. Cytotoxic assays showed that ALL relapses remained sensitive to lysis mediated either by ADCC, CAR-T, or TCR, even if the lysis kinetics were different depending on the effector used. We also identified an immunosuppressive monocytic population in the last relapse sample that may have led to low persistence of CAR-T. CONCLUSION: CD19+ relapses of ALL remain sensitive to cell lysis mediated by T-cell effectors. In case of ALL relapses after immunotherapy, a large immunophenotype will make new therapies possible for controlling such high risk ALL.
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T-cell receptors (TCRs) are formed by stochastic gene rearrangements, theoretically generating >1019 sequences. They are selected during thymopoiesis, which releases a repertoire of about 108 unique TCRs per individual. How evolution shaped a process that produces TCRs that can effectively handle a countless and evolving set of infectious agents is a central question of immunology. The paradigm is that a diverse enough repertoire of TCRs should always provide a proper, though rare, specificity for any given need. Expansion of such rare T cells would provide enough fighters for an effective immune response and enough antigen-experienced cells for memory. We show here that human thymopoiesis releases a large population of clustered CD8+ T cells harboring α/ß paired TCRs that (i) have high generation probabilities and (ii) a preferential usage of some V and J genes, (iii) which CDR3 are shared between individuals, and (iv) can each bind and be activated by multiple unrelated viral peptides, notably from EBV, CMV, and influenza. These polyspecific T cells may represent a first line of defense that is mobilized in response to infections before a more specific response subsequently ensures viral elimination. Our results support an evolutionary selection of polyspecific α/ß TCRs for broad antiviral responses and heterologous immunity.
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Antígenos Virales , Linfocitos T CD8-positivos , Humanos , Antígenos Virales/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T/genética , PéptidosRESUMEN
Human papillomavirus (HPV) infections account for more than 50% of infection-linked cancers in women worldwide. The immune system controls, at least partially, viral infection and around 90% of HPV-infected women clear the virus within two years. However, it remains unclear which immune cells are implicated in this process and no study has evaluated the direct interaction between HPVs and NK cells, a key player in host resistance to viruses and tumors. We demonstrated an NK-cell infiltration in HPV-associated preneoplastic cervical lesions. Since HPVs cannot grow in vitro, virus-like particles (VLPs) were used as a model for studying the NK-cell response against the virus. Interestingly, NK cells displayed higher cytotoxic activity and cytokine production (TNF-α and IFN-γ) in the presence of HPV-VLPs. Using flow cytometry and microscopy, we observed that NK-cell stimulation was linked to rapid VLP entry into these cells by macropinocytosis. Using CD16(+) and CD16(-) NK-cell lines and a CD16-blocking antibody, we demonstrated that CD16 is necessary for HPV-VLP internalization, as well as for degranulation and cytokine production. Thus, we show for the first time that NK cells interact with HPVs and can participate in the immune response against HPV-induced lesions.
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Carcinoma de Células Escamosas/virología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/virología , Papillomaviridae/inmunología , Receptores de IgG/inmunología , Neoplasias del Cuello Uterino/virología , Western Blotting , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Separación Celular , Citocinas/metabolismo , Femenino , Humanos , Inmunoprecipitación , Células Asesinas Naturales/metabolismo , Microscopía Confocal , Infecciones por Papillomavirus/inmunología , Lesiones Precancerosas/inmunología , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/virología , Receptores de IgG/biosíntesis , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/metabolismo , Internalización del Virus , Displasia del Cuello del Útero/inmunología , Displasia del Cuello del Útero/metabolismo , Displasia del Cuello del Útero/virologíaRESUMEN
We exploited traceable gene tagging in primary human T cells to establish the composition and dynamics of seven canonical TCR-induced protein signaling complexes (signalosomes) using affinity purification coupled with mass spectrometry (AP-MS). It unveiled how the LAT adaptor assembles higher-order molecular condensates and revealed that the proximal TCR-signaling network has a high degree of qualitative and quantitative conservation between human CD4+ and CD8+ T cells. Such systems-level conservation also extended across human and mouse T cells and unexpectedly encompassed protein-protein interaction stoichiometry. Independently of evolutionary considerations, our study suggests that a drug targeting the proximal TCR signaling network should behave similarly when applied to human and mouse T cells. However, considering that signaling differences likely exist between the distal TCR-signaling pathway of human and mouse, our fast-track AP-MS approach should be favored to determine the mechanism of action of drugs targeting human T cell activation. An opportunity is illustrated here using an inhibitor of the LCK protein tyrosine kinase as a proof-of-concept.
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Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Biomarcadores , Comunicación Celular/inmunología , Edición Génica , Humanos , Inmunofenotipificación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Modelos Biológicos , Fosforilación , Mapeo de Interacción de Proteínas , Especificidad de la Especie , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
A full exploration of immune responses is deserved after anti-SARS-CoV-2 vaccination and boosters, especially in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although several reports indicate successful humoral responses in such patients, the literature is scarce on cellular specific immunity. Here, both B- (antibodies) and T-cell responses were explored after one (V3 n = 40) or two (V4 n = 12) BNT162b2 mRNA vaccine boosters in 52 allo-HSCT recipients at a median of 755 days post-transplant (<1 year n = 9). Results were compared with those of 12 controls who had received only one booster (BNT162b2 n = 6; mRNA-1273 n = 6). All controls developed protective antibody levels (>250 BAU/mL) and anti-spike T-cell responses. Similarly, 81% of the patients developed protective antibody levels, without difference between V3 and V4 (82.5% vs. 75%, p = 0.63), and 85% displayed T-cell responses. The median frequency of anti-spike T cells did not differ either between controls or the whole cohort of patients, although it was significantly lower for V3 (but not V4) patients. COVID-19 infections were solely observed in individuals having received only one booster. These results indicate that four vaccine injections help to achieve a satisfactory level of both humoral and cellular immune protection in allo-HSCT patients.
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COVID-19 , Trasplante de Células Madre Hematopoyéticas , Vacunas , Vacuna BNT162 , COVID-19/prevención & control , Vacunas contra la COVID-19 , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas , Humanos , Inmunización Secundaria , SARS-CoV-2 , Linfocitos T , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
BACKGROUND: At variance to humoral responses, cellular immunity after anti-SARS-CoV-2 vaccines has been poorly explored in recipients of allogeneic hematopoietic stem-cell transplantation (Allo-HSCT), especially within the first post-transplant years where immunosuppression is more profound and harmful. METHODS: SARS-CoV-2 Spike protein-specific T-cell responses were explored after two doses of BNT162b2 mRNA vaccine in 45 Allo-HSCT recipients with a median time from transplant of less than 2 years by using INF-γ ELISPOT assay and flow-cytometry enumeration of CD4+ and CD8+ T lymphocytes with intracellular cytokine production of IFN-γ and TNF-α. RESULTS: A strong TNF-α+ response from SARS-CoV-2-specific CD4+ T-cells was detected in a majority of humoral responders (89%) as well as in a consistent population of non-humoral responders (40%). CONCLUSIONS: T-cells are likely to participate in protection against COVID-19 viral infection, even in the absence of detectable antibody response, especially in the first years post-transplant in Allo-HSCT recipients.
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The impact of pre-transplant anti-severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine in 20 recipients of allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and/or their donors is reported here, showing that the persistence of anti-SARS-CoV-2 antibodies can be detected in almost all patients, whatever the type of vaccine used, and up to 9 months post transplant. Also, an anti-SARS-CoV-2 spike glycoprotein CD3+ T-cell response could be detected in six (35%) of 17 evaluable patients. This study provides a rationale to consider anti-SARS-CoV-2 vaccination of both recipients and donors before Allo-HSCT.
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Intercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcgammaR as well as the associated FcRgamma subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcgammaR-expressing APC. Capture of FcgammaR occurred during coculture of T cells with FcgammaR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcgammaR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcgammaR captured by T cells, and biochemical studies suggested the improper integration of FcgammaR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcgammaR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation.
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Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Receptores de IgG/inmunología , Animales , Presentación de Antígeno/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Calcio/inmunología , Calcio/metabolismo , Comunicación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ligandos , Ratones , Ratones Transgénicos , Muramidasa/inmunología , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Receptores de IgG/metabolismo , Transducción de Señal/inmunología , TransfecciónRESUMEN
Multiple myeloma (MM) is an incurable cancer characterized by the proliferation and accumulation of monoclonal plasma cells in the bone marrow. The monoclonal anti-CD38 daratumumab has taken a central place in the different treatment regimens for newly diagnosed and relapsed, refractory myeloma. In this study, we correlated the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and potential fratricide induced by daratumumab with CD38-expression levels on both effector and target cells. We show that CD38 expression can be modulated by adding all-trans retinoic acid (ATRA) or interferon-α to MM cells to further fine-tune these effects. In addition, we observed that ADCC becomes inefficient when fratricide occurs and both ADCC and fratricide depend on the balance between CD38 expression on effector and target cells. However, the addition of adjuvants (retinoic acid or interferon-α) to myeloma cells or the inhibition of fratricide using a CD38-blocking nanobody on NK-cells can reverse this balance towards ADCC and thus promote lysis of target cells by ADCC. ATRA and interferon-α increased the CD38 expression at the surface of MM cells about three-fold and two-fold, respectively. This increase was of interest for MM cells with low CD38 expression, that became susceptible to daratumumab-mediated ADCC after preincubation. A CD38-blocking nanobody prevented the binding of daratumumab to these NK-cells and blunted the fratricidal effect on effector NK cells. In conclusion, our study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated ADCC.
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Malignant central nervous system tumors are the leading cause of cancer death in children. Progress in high-throughput molecular techniques has increased the molecular understanding of these tumors, but the outcomes are still poor. Even when efficacious, surgery, radiation, and chemotherapy cause neurologic and neurocognitive morbidity. Adoptive cell therapy with autologous CD19 chimeric antigen receptor T cells (CAR T) has demonstrated remarkable remission rates in patients with relapsed refractory B cell malignancies. Unfortunately, tumor heterogeneity, the identification of appropriate target antigens, and location in a growing brain behind the blood-brain barrier within a specific suppressive immune microenvironment restrict the efficacy of this strategy in pediatric neuro-oncology. In addition, the vulnerability of the brain to unrepairable tissue damage raises important safety concerns. Recent preclinical findings, however, have provided a strong rationale for clinical trials of this approach in patients. Here, we examine the most important challenges associated with the development of CAR T cell immunotherapy and further present the latest preclinical strategies intending to optimize genetically engineered T cells' efficiency and safety in the field of pediatric neuro-oncology.
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The antibody-dependent cellular cytotoxicity (ADCC) effector function of natural killer (NK) cells is one of the known mechanisms of action for rituximab-based anti-cancer immunotherapy. Inhibition of the ADCC function of NK cells through interactions between inhibitory killer cell immunoglobulin-like receptors (KIRs) and HLA class I ligands is associated with resistance of cancers to rituximab. In this study, we deeply investigated the impact of KIR, HLA class I, and CD16 genotypes on rituximab-dependent NK cell responses in both an in vitro cellular model from healthy blood donors and ex vivo rituximab-treated non-Hodgkin lymphoma (NHL) patients. We highlight that an HLA environment with limited KIR ligands is beneficial to promoting a higher frequency of KIR+ NK cells including both educated and uneducated NK cells, two NK cell compartments that demonstrate higher rituximab-dependent degranulation than KIR- NK cells. In contrast, a substantial KIR ligand environment favors a higher frequency of poorly effective KIR- NK cells and numerous functional KIR/HLA inhibitions of educated KIR+ NK cells. These phenomena explain why NHL patients with limited KIR ligands respond better to rituximab. In this HLA environment, CD16 polymorphism appears to have a collateral effect. Furthermore, we show the synergic effect of KIR2DS1, which strongly potentiates NK cell ADCC from C2- blood donors against C2+ target cells. Taken together, these results pave the way for stronger prediction of rituximab responses for NHL patients. HLA class I typing and peripheral blood KIR+ NK cell frequency could be simple and useful markers for predicting rituximab response.
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Células Asesinas Naturales , Linfoma , Receptores KIR , Rituximab , Citotoxicidad Celular Dependiente de Anticuerpos , Humanos , Ligandos , Rituximab/farmacologíaRESUMEN
BACKGROUND: Susceptibility to Covid-19 has been found to be associated with the ABO blood group, with O type individuals being at a lower risk. However, the underlying mechanism has not been elucidated. Here, we aimed to test the hypothesis that Covid-19 patients might have lower levels of ABO antibodies than non-infected individuals as they could offer some degree of protection. METHODS: After showing that the viral spike protein harbors the ABO glycan epitopes when produced by cells expressing the relevant glycosyltransferases, like upper respiratory tract epithelial cells, we enrolled 290 patients with Covid-19 and 276 asymptomatic controls to compare their levels of natural ABO blood group antibodies. RESULTS: We found significantly lower IgM anti-A + anti-B agglutination scores in blood group O patients (76.93 vs 88.29, P-value = 0.034) and lower levels of anti-B (24.93 vs 30.40, P-value = 0.028) and anti-A antibodies (28.56 vs 36.50, P-value = 0.048) in blood group A and blood group B patients, respectively, compared to controls. CONCLUSION: In this study, we showed that ABO antibody levels are significantly lower in Covid-19 patients compared to controls. These findings could indicate that patients with low levels of ABO antibodies are at higher risk of being infected.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Anticuerpos/sangre , COVID-19/sangre , Polisacáridos/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/virología , Susceptibilidad a Enfermedades , Células Epiteliales/inmunología , Epítopos/inmunología , Femenino , Galactosiltransferasas , Humanos , Inmunoglobulina M/inmunología , Masculino , Persona de Mediana Edad , Riesgo , Adulto JovenRESUMEN
Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) are used to prepare human EBV-specific T lymphocytes (EBV-CTL) in vitro. Within an LCL, up to 5-7% the cells release infectious EBV, and this has fostered safety concerns for therapeutic applications because of the exposure of T cells to EBV. The release of infectious viruses can be prevented by ganciclovir, but this drug may seriously affect LCL growth. In the wake of these concerns, the present work was designed to compile safety data on EBV-CTL preparation for the purpose of submission to a regulatory agency. We showed that further to supernatant exclusion, the number of EBV genome copies (EBVc) associated with the EBV-CTL always made up a constant proportion of the EBVc number detected in the culture supernatant. In addition, such was the case whether infectious virus could be produced by the LCL or not, suggesting that the EBV signal detected was due to a DNA contamination rather than an infection. Furthermore, we demonstrated that the number of EBVc associated with the EBV-CTL was highly sensitive to DNAse treatment, and finally that EBVc could no longer be detected after the EBV-CTL had been amplified in the absence of LCL. Consequently, during in vitro EBV-CTL preparation, either T cells cannot be infected or they die rapidly after EBV infection.