RESUMEN
Patients with chronic lymphocytic leukemia (CLL) or monoclonal B-lymphocytosis (MBL) have impaired response to COVID-19 vaccination. A total of 258 patients (215 with CLL and 43 with MBL) had antispike antibody levels evaluable for statistical analysis. The overall seroconversion rate in patients with CLL was 94.2% (antispike antibodies ≥50 AU/mL) and 100% in patients with MBL after multiple vaccine doses. After 3 doses (post-D3) in 167 patients with CLL, 73.7% were seropositive, 17.4% had antispike antibody levels between 50 and 999 AU/mL, and 56.3% had antispike antibody levels ≥1000 AU/mL, with a median rise from 144.6 to 1800.7 AU/mL. Of patients who were seronegative post-D2, 39.7% seroconverted post-D3. For those who then remained seronegative after their previous dose, seroconversion occurred in 40.6% post-D4, 46.2% post-D5, 16.7% post-D6, and 0% after D7 or D8. After seroconversion, most had a progressive increase in antispike antibody levels. Neutralization was associated with higher antispike antibody levels, more vaccine doses, and earlier severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants; neutralizing antibody against early clade D614G was detected in 65.3%, against Delta in 52.0%, and against Omicron in 36.5%. SARS-CoV-2-specific T-cell production of interferon γ and interleukin 2 occurred in 73.9% and 60.9%, respectively, of 23 patients tested. After multiple vaccine doses, by multivariate analysis, immunoglobulin M ≥0.53 g/L, immunoglobulin subclass G3 ≥0.22 g/L and absence of current CLL therapy were independent predictors of positive serological responses. Multiple sequential COVID-19 vaccination significantly increased seroconversion and antispike antibody levels in patients with CLL or MBL.
Asunto(s)
COVID-19 , Leucemia Linfocítica Crónica de Células B , Linfocitosis , Humanos , Vacunas contra la COVID-19 , Leucemia Linfocítica Crónica de Células B/terapia , Seroconversión , COVID-19/prevención & control , SARS-CoV-2 , Inmunoglobulina M , Inmunoglobulina G , Inmunidad , Anticuerpos AntiviralesRESUMEN
We developed a 25-color flow cytometry panel to comprehensively interrogate innate lymphoid cells (ILC), mucosal-associated invariant T (MAIT) cells, natural killer (NK) cells and γδ T cells in human tissues. The ability to isolate and interrogate these cells from fresh human tissue is crucial in understanding the role these cells play at immune-privileged mucosal surfaces like the intestine in health and disease settings. However, liberating these cells from tissue is extremely challenging as many key surface identification markers are susceptible to enzymatic cleavage. Choosing the correct enzyme-antibody clone combination within a high-parameter panel is, therefore, a critical consideration. Here, we present a comprehensive, in-depth analysis of the effect different common digestive enzyme blends have on key surface markers used to identify these cell types. In addition, we compared multiple antibody clones for surface markers that are highly susceptible to enzymatic cleavage, such as CD127 and NKp44, to achieve the most consistent and superior staining patterns among donors.
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Células T Invariantes Asociadas a Mucosa , Biomarcadores , Citometría de Flujo , Humanos , Inmunidad Innata , Intestinos , Células Asesinas NaturalesRESUMEN
Identifying SARS-CoV-2-specific T cell epitope-derived peptides is critical for the development of effective vaccines and measuring the duration of specific SARS-CoV-2 cellular immunity. In this regard, we previously identified T cell epitope-derived peptides within topologically and structurally essential regions of SARS-CoV-2 spike and nucleocapsid proteins by applying an immunoinformatics pipeline. In this study, we selected 30 spike- and nucleocapsid-derived peptides and assessed whether these peptides induce T cell responses and avoid major mutations found in SARS-CoV-2 variants of concern. Our peptide pool was highly specific, with only a single peptide driving cross-reactivity in people unexposed to SARS-COV-2, and immunogenic, inducing a polyfunctional response in CD4+ and CD8+ T cells from COVID-19 recovered individuals. All peptides were immunogenic and individuals recognized broad and diverse peptide repertoires. Moreover, our peptides avoided most mutations/deletions associated with all four SARS-CoV-2 variants of concern while retaining their physicochemical properties even when genetic changes are introduced. This study contributes to an evolving definition of individual CD4+ and CD8+ T cell epitopes that can be used for specific diagnostic tools for SARS-CoV-2 T cell responses and is relevant to the development of variant-resistant and durable T cell-stimulating vaccines.
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COVID-19 , SARS-CoV-2 , Humanos , Epítopos de Linfocito T , PéptidosRESUMEN
BACKGROUND: Intestinal neutrophil recruitment is a characteristic feature of the earliest stages of inflammatory bowel disease (IBD). Neutrophil elastase (NE) and myeloperoxidase (MPO) mediate the formation of neutrophil extracellular traps (NETs); NETs produce the bactericidal oxidant hypochlorous acid (HOCl), causing host tissue damage when unregulated. The project aim was to investigate the relationship between NET formation and clinical IBD in humans. METHODS: Human intestinal biopsies were collected from Crohn's disease (CD) patients, endoscopically categorized as unaffected, transitional, or diseased, and assigned a histopathological score. RESULTS: A significant linear correlation was identified between pathological score and cell viability (TUNEL+). Immunohistochemical analysis revealed the presence of NET markers NE, MPO, and citrullinated histone (CitH3) that increased significantly with increasing histopathological score. Diseased specimens showed greater MPO+-immunostaining than control (P < .0001) and unaffected CD (P < .0001), with transitional CD specimens also showing greater staining than controls (P < .05) and unaffected CD (P < .05). Similarly, NE+-immunostaining was elevated significantly in diseased CD than controls (P < .0001) and unaffected CD (P < .0001) and was significantly higher in transitional CD than in controls (P < .0001) and unaffected CD (P < .0001). The CitH3+-immunostaining of diseased CD was significantly higher than controls (P < .05), unaffected CD (P < .0001) and transitional CD (P < .05), with transitional CD specimens showing greater staining than unaffected CD (P < .01). Multiplex immunohistochemistry with z-stacking revealed colocalization of NE, MPO, CitH3, and DAPI (cell nuclei), confirming the NET assignment. CONCLUSION: These data indicate an association between increased NET formation and CD severity, potentially due to excessive MPO-mediated HOCl production in the extracellular domain, causing host tissue damage that exacerbates CD.
Our data show for the first time that the density of neutrophil extracellular trap formed in the bowel of Crohn's disease patients increases with increasing disease severity, suggesting that myeloperoxidase-mediated host-tissue damage may play a role in disease pathogenesis.
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Enfermedad de Crohn , Trampas Extracelulares , Enfermedad de Crohn/patología , Trampas Extracelulares/metabolismo , Histonas , Humanos , Infiltración Neutrófila , Neutrófilos/metabolismo , Peroxidasa/metabolismoRESUMEN
The human intestine contains numerous mononuclear phagocytes (MNP), including subsets of conventional dendritic cells (cDC), macrophages (Mf) and monocytes, each playing their own unique role within the intestinal immune system and homeostasis. The ability to isolate and interrogate MNPs from fresh human tissue is crucial if we are to understand the role of these cells in homeostasis, disease settings and immunotherapies. However, liberating these cells from tissue is problematic as many of the key surface identification markers they express are susceptible to enzymatic cleavage and they are highly susceptible to cell death. In addition, the extraction process triggers immunological activation/maturation which alters their functional phenotype. Identifying the evolving, complex and highly heterogenous repertoire of MNPs by flow cytometry therefore requires careful selection of digestive enzyme blends that liberate viable cells and preserve recognition epitopes involving careful selection of antibody clones to enable analysis and sorting for functional assays. Here we describe a method for the anatomical separation of mucosa and submucosa as well as isolating lymphoid follicles from human jejunum, ileum and colon. We also describe in detail the optimised enzyme digestion methods needed to acquire functionally immature and biologically functional intestinal MNPs. A comprehensive list of screened antibody clones is also presented which allows for the development of high parameter flow cytometry panels to discriminate all currently identified human tissue MNP subsets including pDCs, cDC1, cDC2 (langerin+ and langerin-), newly described DC3, monocytes, Mf1, Mf2, Mf3 and Mf4. We also present a novel method to account for autofluorescent signal from tissue macrophages. Finally, we demonstrate that these methods can successfully be used to sort functional, immature intestinal DCs that can be used for functional assays such as cytokine production assays.
Asunto(s)
Separación Celular , Colon/citología , Citometría de Flujo , Íleon/citología , Mucosa Intestinal/citología , Yeyuno/citología , Fagocitos/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Fagocitos/inmunología , FenotipoRESUMEN
Langerhans cells (LC) are thought to be the only mononuclear phagocyte population in the epidermis where they detect pathogens. Here, we show that CD11c+ dendritic cells (DCs) are also present. These cells are transcriptionally similar to dermal cDC2 but are more efficient antigen-presenting cells. Compared to LCs, epidermal CD11c+ DCs are enriched in anogenital tissues where they preferentially interact with HIV, express the higher levels of HIV entry receptor CCR5, support the higher levels of HIV uptake and replication and are more efficient at transmitting the virus to CD4 T cells. Importantly, these findings are observed using both a lab-adapted and transmitted/founder strain of HIV. We also describe a CD33low cell population, which is transcriptionally similar to LCs but does not appear to function as antigen-presenting cells or acts as HIV target cells. Our findings reveal that epidermal DCs in anogenital tissues potentially play a key role in sexual transmission of HIV.