Immunophenotyping Of Leukocytes Research Articles

42-parameter mass cytometry panel to assess cellular and functional phenotypes of leukocytes in bronchoalveolar lavage of Rhesus macaque.

This Optimized Multiparameter Immunofluorescence Panel (OMIP) reports on the development of a mass cytometry panel for broad immunophenotyping of leukocytes from bronchoalveolar lavage from rhesus macaques. Using this panel, we were able to identify myeloid populations such as macrophages, neutrophils, monocytes, myeloid and plasmacytoid DCs, basophils and lymphoid cell lineages including B cells, natural killer (NK) cells, mucosal associated invariant T (MAIT) cells, γδ T cells, CD4 T cells, CD8□β T cells, CD8 □□ T cells, and innate lymphoid cells (ILCs). We also included markers for defining memory, differentiation (CCR7, CD28, CD45RA), homing potential (CXCR3), cytotoxic potential (perforin, granzyme B, granzyme K), cell activation/differentiation (HLA-DR, CD69, IgD) and effector function (CD154, IFN-γ, TNF, IL-2, IL-17A, IL-6, IL-1β, CCL4 and CD107a). This panel was optimized on cryopreserved, bronchoalveolar lavage and splenocytes collected from rhesus macaques. The antibodies selected in this panel are human-specific antibodies that have been shown to cross-react with non-human primates except for CD45 clone D058-1283 which is specific for non-human primates.

Effect of Immunity to SARS-CoV-2 Virus on Blood Cellular Composition

Relevance. The new coronavirus infection (COVID-19) is still a public health problem and a threat to socio-economic well-being. Most studies have focused predominantly on humoral immunity, and there are no data on the cellular composition of blood in dynamics. Aim. To study the dynamics of changes in blood cellular composition depending on the type of immunity formed (natural, hybrid, breakthrough, postvaccinal) to SARS-CoV-2 virus. Materials and Methods. A total of 130 volunteers participated in the study. Immunophenotyping of peripheral blood leukocytes using flow cytometry was performed. The presence of specific IgG antibodies to N-protein SARS-CoV-2, total IgA and cytokines (IL-4, IL-10, IFN-γ, TNF-α) was assessed in serum by ELISA. Results and Discussion. A statistically significant increase in BL was recorded in volunteers with hybrid immunity 1 month (14,0% (12,3–16,4%)) after vaccination compared to healthy volunteers (9,1% (6,4–10,2%), p = 0,0007) and people with primary COVID-19 infection (10,2% (8,3–12,1%), p = 0,0134). In volunteers with natural and hybrid immunity, as well as in revaccinated people, an increase in B1-cells (CD3-CD19+CD5+CD27-) was observed during 3–9 months of observation. It is shown that the increase of B-lymphocytes with «switched» class of synthesized antibodies was detected in people with breakthrough immunity. Increased levels of T-lymphocytes expressing HLA-DR were recorded in all individuals during 6–9 months of follow-up. Volunteers with breakthrough immunity showed a significant increase in the positivity index when assessing the presence of specific IgG class antibodies to the coronavirus N-protein compared with volunteers with natural and hybrid immunity. Conclusions. Vaccination promotes protective immunity sufficient for timely activation of memory T- and B-cells in breakthrough immunity and maintenance of immunologic efficacy in hybrid immunity against COVID-19. The results help to assess the strain of innate and adaptive immunity in novel coronavirus infection and to fill gaps in the understanding of immunopathogenesis in COVID-19.

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Immunogenicity and efficacy of an oral live-attenuated vaccine for bovine Johne's disease.

Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of Johne's disease (JD) in ruminants, establishes a prolonged and often lifelong enteric infection. The implementation of control measures for bovine JD has faced obstacles due to the considerable expenses involved in disease surveillance and hindered by unreliable and inadequate diagnostic tests, emphasizing the need for an effective vaccine that can stimulate mucosal immunity in the gastrointestinal tract. Previous investigations have demonstrated that deletion of the BacA gene in MAP produces an attenuated strain that can transiently colonize the calf small intestine while retaining its capacity to stimulate systemic immune responses similar to wildtype MAP strains. This study assessed the efficacy of the BacA gene deletion MAP strain, referred to as the BacA vaccine, when administered orally to young calves. The research aimed to evaluate its effectiveness in controlling MAP intestinal infection and to investigate the immune responses elicited by mucosal vaccination. The study represents the first evaluation of an enteric modified live MAP vaccine in the context of an oral MAP challenge in young calves. Oral immunization with BacA reduced MAP colonization specifically in the ileum and ileocecal valve. This partially protective immune response was associated with an increased frequency of CD4+ and CD8+ T cells with a pro-inflammatory phenotype (IFNγ+/TNFα+) in vaccinated animals. Moreover, re-stimulated PBMCs from vaccinated animals showed increased expression of IFNγ, IP-10, IL-2, and IL-17 at 10- and 12-weeks post challenge. Furthermore, immunophenotyping of blood leukocytes revealed that vaccinated calves had increased levels of T cells expressing cell-surface markers consistent with long-term central memory. Overall, our findings provide new insights into the development and immunogenicity of a modified live MAP vaccine against bovine JD, demonstrating oral vaccination can stimulate host immune responses that can be protective against enteric MAP infection.

Immunophenotyping of Leukocytes in Amniotic Fluid.

Immunophenotyping allows forthe deep characterization of leukocytes present in biological samples. Here, we describe a complete procedure for the immunophenotyping of amniotic fluid, which can provide information into the immune processes taking place in the amniotic cavity. The protocol describes amniotic fluid cell count determination, processing, and the use of viability, extracellular antibody, and intracellular/intranuclear antibody staining prior to flow cytometer acquisition.

Isolation and Immunophenotyping of Leukocytes from the Human Maternal-Fetal Interface.

During human pregnancy, leukocytes that infiltrate the maternal-fetal interface play a major role in establishing a delicate balance between immune tolerance and functional response and setting the inflammatory process that leads to labor. Here we describe two methods for isolating immune cells from the chorioamniotic membranes (decidua parietalis) and placental blood (decidua basalis) that combine gentle enzymatic digestion, magnetic cell sorting, and density gradient. Isolated leukocytes can be immunophenotypified by flow cytometry, and both isolation methods are compatible with downstream cellular and molecular applications, such as cell culture, transcriptome, and proteome analyses.

Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy.

The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay.

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/μL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.

Hybrid Macromolecular Constructs as a Platform for Spectral Nanoprobes for Advanced Cellular Barcoding in Flow Cytometry.

Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.

Assessment of changes in the phenotype, intensity of degranulation, death and lysis of neutrophils in &lt;i&gt;ex vivo&lt;/i&gt; modeling of &lt;i&gt;Staphylococcal&lt;/i&gt; bacteremia

Introduction. At present, it is important to study the interaction of Staphylococcus aureus with human innate immunity cells under conditions of ex vivo bacteremia modeling, but not only in experiments with phagocytes previously isolated from the blood.&#x0D; Purpose: comparative assessment of changes in the phenotype, intensity of aurophilic degranulation, death and lysis of neutrophils in whole blood samples from apparently healthy donors following the addition of live and killed microbial cells of S. aureus ATCC 6538 (209-P).&#x0D; Materials and methods. Bacteremia was modeled by adding microbes to whole blood (with heparin) at doses ranging from 108 to 101 mc/ml. The bactericidal effect was determined at different time points of blood incubation at 37C (for 6 hours) by the microbiological method. The blood leukocyte immunophenotyping was performed using flow cytometry according to the Lyse/No-Wash protocol to determine the expression of markers of azurophilic degranulation (CD63), early apoptosis (CD95) on the surface of neutrophilic granulocytes (NG) and cell adhesion (CD62L), as well as to assess the surface expression density of the molecular trigger of netosis FcᵧRIIIb (CD16) and tyrosine protein phosphatase (CD45), which is a regulator of FcR-mediated cell signaling during phagocytosis and netosis. The presence of degenerative changes in NG was confirmed by microscopy in the analysis of blood smears stained according to RomanovskyGiemsa.&#x0D; Results. Significant differences were revealed in the response of human blood NG to live and killed S. aureus cells according to the studied phenotypic parameters. Only live microbes triggered azurophilic degranulation in NCs and stimulated, starting from 2 h incubation, the appearance of a large number of NG with the CD95+ phenotype in the blood. In addition, live bacteria induced in phagocytes twice as much functional rearrangement in terms of CD45 expression. They induced a rapid decrease in the density of CD16 expression on NG and activated NG lysis in the blood with an intensity depending on the initial microbial concentration. Following 6 h incubation at concentrations of more than 105 mc/ml, flow cytometry detected the presence in the blood of a population of low density neutrophils characteristic of sepsis (Low Density Neutrophils with the CD63+CD16low phenotype), whose cells are prone to spontaneous autolysis (netosis).&#x0D; Conclusion. Under the conditions of ex vivo modeling of bacteremia, various strategies of human blood neutrophils in the implementation of the mechanisms of protection of the macroorganism from infection were evaluated, and the prospects of using the obtained information to identify individuals at high risk of developing staphylococcal sepsis were determined.

Obesity in Severe COVID-19 Patients Has a Distinct Innate Immune Phenotype.

Obesity alters the capacity of effective immune responses in infections. To further address this phenomenon in the context of COVID-19, this study investigated how the immunophenotype of leukocytes was altered in individuals with obesity in severe COVID-19. This cross-sectional study enrolled 27 ICU COVID-19 patients (67% women, 56.33 ± 19.55 years) that were assigned to obese (BMI ≥ 30 kg/m2, n = 9) or non-obese (BMI < 30kg/m2, n = 18) groups. Monocytes, NK, and both Low-Density (LD) and High-Density (HD) neutrophils were isolated from peripheral blood samples, and surface receptors' frequency and expression patterns were analyzed by flow cytometry. Clinical status and biochemical data were additionally evaluated. The frequency of monocytes was negatively correlated with BMI, while NK cells and HD neutrophils were positively associated (p < 0.05). Patients with obesity showed a significant reduction of monocytes, and these cells expressed high levels of PD-L1 (p < 0.05). A higher frequency of NK cells and increased expression of TREM-1+ on HD neutrophils were detected in obese patients (p < 0.05). The expression of receptors related to antigen-presentation, phagocytosis, chemotaxis, inflammation and suppression were strongly correlated with clinical markers only in obese patients (p < 0.05). Collectively, these outcomes revealed that obesity differentially affected, and largely depressed, innate immune response in severe COVID-19.

Comparative characteristics of the cellular immune response to SARS-CoV-2 during infection and post-vaccination

An important area of research concerns monitoring of immune response features in patients with SARS-CoV-2 infection as well as their analysis, as compared with characteristics of vaccine-mediated protection, in order to specify the determinants of cellular immune response. The aim of our work was to compare the state of cellular immune response in patients who underwent COVID-19, and in persons vaccinated with a peptide vaccine preparation. The study involved volunteers who suffered with COVID-19 of varying severity (n = 30), as well as persons who completed the full course of vaccination with the peptide vaccine (n = 27). For comparison, we took blood specimens from the volunteers before vaccination. Immunophenotyping of leukocytes was performed by the Lyse/No-Wash procedure (BD Bioscience, USA), and Cyto-Stat monoclonal antibodies (CD45-FITC, CD4-PE, CD8-ECD, CD3-PC5), CD45RA-PC7, CD45RO-PE (Beckman Coulter, USA), and analyzed with a DakoCytomation flow cytometer (Denmark). Determination of intracellular IFNγ (CD4+IFNγ+) was performed with the standard technique. Cytokine production was determined using reagent kits for detection of IFNγ, TNFα, IL-4, IL-8, IL-10 (Vector-Best JSC, Russia) with automatic enzyme immunoassay analyzer LAZURIT (Dynex Technologies, USA). As based on the results obtained, we have shown that cellular immunity was developed after vaccination and infection with COVID-19. However, the most pronounced immune response was recorded in the COVID-19 reconvalescents, i.e., more than 60% of these patients showed an increased number of CD4+T-memory helper cells (8.7 (0.5-12.1) % versus 0.3 (0.1-0.5) % in the comparison group, p &lt; 0.05) as well as proportion of CD4+IFNγ+T lymphocytes (4.2 (1.8-4.3) % versus 0.4 (0-0.8) % in the comparison group, p &lt; 0.05). Moreover, we revealed an increased functional reserve of cells in terms of TNFα, IL-8, IL-10 production. One month after vaccination of volunteers with the peptide-based preparation, the total pool of memory T lymphocytes was apparently dominated by CD8+T memory cells (CD45+CD8+CD45RA-CD45RO+). A significant increase was found in the average levels of CD4+IFNγ+ activated cells (8.2-fold), as well as in values of ConA-induced IL-4 production (3.3 (1.1-4.5) pg/mL, and 2.8 (1.7-3.9) pg/mL, respectively versus 1.3 (0.1-2.4) pg/mL in the control group, p &lt; 0.05). The data obtained are in accordance with information available in the literature concerning development of cellular immune responses to SARS-CoV-2, which results from a past illness, or measures for the specific prevention of COVID-19. Further search for cellular correlates of protection against a new coronavirus infection will allow us to revise the current vaccination strategy and develop an optimal approach to COVID-19 prevention.

Increased expression of tyrosine protein phosphatase (CD45) on the surface of human blood granulocytes under the influence of the plague microbe vaccine strain &lt;I&gt;Yersinia pestis&lt;/I&gt; EV NIIEG &lt;I&gt;ex vivo&lt;/I&gt; and &lt;I&gt;in vivo&lt;/I&gt;

Tyrosine protein phosphatase (common leukocyte antigen CD45) regulates FcᵧR-mediated cell signaling and secretory function of neutrophilic granulocytes (NG) when interacting with antigen-antibody immune complexes. The aim of the work is to study changes in the expression of CD45 on the surface of human granulocytes in ex vivo modeling of bacteremia by live cells of the plague microbe vaccine strain Yersinia pestis EV NIIEG and to evaluate the priming effect of the live plague vaccine (LPV) in vivo in terms of this parameter. The expression density of CD45 on NG was determined by flow cytometry in conventional units of fluorescence intensity (MFI) after staining the cells with the CD45-FITC labeled mouse antibody reagent (Backman Coulter, USA) during immunophenotyping of blood leukocytes according to the Lyse/No Wash protocol. In donors not previously vaccinated against plague (group 1), the value of the indicator was assessed before and 30 min, 2 h, 6 h after the addition of Y pestis EV cells to whole blood at a dose of 108 mc/ml, as well as 1 month and 6 months after the primary anti-plague vaccination. In individuals who had previously been repeatedly vaccinated with LPV in the territory of the natural plague focus (group 2), CD45 expression on blood granulocytes was determined one year after the last annual vaccination, and then 1 month and 6 months after revaccination. Getting into human blood, living cells of the vaccine strain Y pestis EV of the plague microbe induced a change in the NC phenotype already after 30 minutes, associated with a 3.5-fold increase in the surface expression of CD45, which remained at an elevated level for 6 hours. The studied indicator depended ex vivo on the degree of resistance of plague microbes to phagocytosis and killing of NG. Plague vaccination had a similar stimulating effect on human peripheral blood NG in vivo. Under the influence of HPV, CD45 expression increased on blood NG in groups 1 and 2 one month after vaccination, and the changes persisted in volunteers for 6 months. The experimental data obtained in the work may reflect the result of NG priming with lipopolysaccharide and other Y. pestis antigens. The registered functional activation of NG by expression of tyrosine protein phosphatase probably indicates the formation of “immune memory” at the level of innate immunity cells under the influence of LPV, the functioning of which explains the development of a faster and more intense antigen-specific immune response to repeated introduction of the plague vaccine into the body.

Indicators of the immune status in patients with chronic wounds depending on the stage of the infectious process and the structure of the wound microbiota

The results of immunophenotyping of peripheral blood leukocytes were analyzed in 105 patients with chronic wounds ( the main group , wound duration more than 3 weeks ) at various stages of the infectious process : colonization (n=39), critical colonization (n=37) and infection (n=29). The comparison group consisted of 22 patients with acute wounds ( wound duration up to 4 days ), in which microorganisms were not detected in the wounds . In the main group , changes in immunological reactivity were revealed in the form of an increase in the relative content of T- lymphocytes and a decrease in NK cells , lower indicators of early activation markers on T- helpers and T- cytotoxic lymphocytes (CD4+/CD38+/CD3+, CD8+/CD38+/CD3+, CD3+/ CD71+) and HLADR on B- lymphocytes , a high level of expression of late markers of T- cell activation (CD3+HLADR+). In the comparison group , the levels of expression of integrins (CD11a, CD11b, CD11c) on neutrophils and lymphocytes , as well as the transferrin receptor (CD71) on neutrophils , monocytes and lymphocytes were higher than in the main group . An increase in the activity of neutrophils in the form of expression of CD11a molecules was revealed with the progression of the infectious process in a chronic wound , which may be an additional diagnostic sign of the transition from the stage of colonization to the stage of critical colonization and infection . The highest values of CD11a and CD11c on neutrophils were recorded in the presence of S. aureus monocultures in chronic wounds , the lowest values were recorded when P. aeruginosa and A. baumannii monocultures were isolated from wounds .

Immunophenotyping to improve the mechanistic understanding of idiosyncratic drug-induced liver injury: clinical implications and future directions

The late event onset of a fraction of idiosyncratic drug-induced liver injury (DILI) cases and the link observed by genome-wide association studies (GWASs) of certain human leucocyte antigen (HLA) alleles with DILI due to specific drugs support the crucial role of the immune system (both innate and adaptive) in the pathogenesis of DILI. Recent advances in both flow and mass cytometry have allowed the profiling of all major immune cell types in a given sample. Therefore, determining the lymphocyte populations in samples from patients with DILI would facilitate the development of specific biomarkers for DILI diagnosis and prognosis. To date, a few studies have explored the immune landscape in DILI. In a recent study of leukocyte immunophenotyping using flow cytometry from the Spanish DILI Registry, an important role of adaptive immune response in DILI is suggested. DILI patients had significantly higher levels of T helper 1 (Th1) cells and activated helper and cytotoxic T cells than healthy controls. Furthermore, the increased expression of negative immune checkpoints and ligands in DILI patients could reflect a restoration of the immune homeostasis. Differences in the profile of cytokines in DILI patients from the Drug-Induced Liver Injury Network (DILIN) also suggest an involvement of both innate and adaptive immune systems in DILI development and prognosis. Moreover, several studies based on immunophenotyping of liver infiltrates showed a distinctive pattern of cellular infiltrates in patients with immune checkpoint inhibitors (ICIs)-DILI, with lower levels of plasma cells, CD20+ B cells and CD4+ T cells than in autoimmune hepatitis (AIH) patients. These pioneering studies highlight the importance of immunophenotyping for the mechanistic understanding of DILI. In this review, available data on immunophenotyping in DILI are gathered, and the potential clinical applications of cutting-edge, novel immunophenotyping techniques are discussed.

Effects of rapid gravity load changes on immunophenotyping and leukocyte function of human peripheral blood after parabolic flight

One of the biological systems that suffers a physiological de-conditioning in space is the immune system. It is in charge of defending the body against pathogens and other aggressions. The aim of this work was to assess if there are any relevant changes in the aggregation of erythrocytes, cell count, immunophenotyping and functionality after parabolic flight. This effect has been assessed ex vivo using human peripheral blood, which was drawn from the radial vein (n = 6 healthy volunteers) and anticoagulated with heparin and EDTA. Blood samples were split into two aliquots and maintained in two identical thermally isolated boxes; one stayed on the ground whereas the other one was subjected to parabolic flight. The parabolic flight consisted of 15 parabolas performed with a Mudry CAP-10B acrobatic aircraft. Each parabola consists of 8 s of hypogravity preceded and followed by 2 s of hypergravity. Any of the biological parameters measured showed no statistically significant differences, with the exception of monocyte counting. Altered gravity could increase aggregation of red blood cells, as demonstrated by a decrease in the number of single cells after parabolic flight exposure. No counting changes in haemoglobin concentration were observed when comparing the two different groups. Furthermore, potential functional alterations of monocytes and neutrophils cannot be rejected. Although these possible changes could be associated with hypogravity, other factors such as hypergravity and acceleration or deceleration cannot be ruled out. Our findings indicate that, under this specific experimental setup, there was no significant alteration in leukocyte immunophenotyping and functional capacity when using ex vivo blood samples and short exposure to altered gravity.

MHCII Expression on Peripheral Blood Monocytes in Canine Lymphoma: An Impact of Glucocorticoids.

Simple SummaryLoss or decreased expression of human leukocyte antigen—D-related (HLA-DR) on the surface of monocytes is related to the dysfunction of the immune system and was reported in human neoplasia, including lymphoma. Canine lymphoma is frequently presented as a valuable comparative model for studies on human non-Hodgkin’s lymphoma. However, there are no studies on the expression of analogue proteins—MHCII antigens—on monocytes in canine lymphoma. In this study, we have evaluated the changes in the expression of MHCII on monocytes in the blood of dogs with lymphoma before any treatment and in dogs that had previously received glucocorticoids. Glucocorticoids are often used by clinicians as first drugs after diagnosis for immediate health improvement and are known to impact monocyte number. We have shown an increase in the percentage of MHCII− monocytes, regardless of treatment. However, only in dogs that had received glucocorticoids were changes in the proportion of MHCII+ and MHCII− monocytes reflected also by the changes in the number of MHCII− monocytes in the blood, which was significantly higher. Evaluating the changes in canine monocytes might be helpful in the diagnosis of various tumor types, monitoring of the treatment or assessing the immune status of dogs.An increase in the percentage of monocytes with reduced HLA-DR expression and immunosuppressive properties has been reported in numerous human neoplastic diseases, including lymphoma. However, there are no analogous studies on phenotypical variations in the peripheral blood monocytes in dogs with lymphoma. The aim of this study was to determine the difference in the expression of the MHCII molecule on peripheral blood monocytes in dogs with lymphoma before any treatment (NRG) and in dogs that had previously received glucocorticoids (RG) in comparison to healthy dogs. Flow cytometry immunophenotyping of peripheral blood leukocytes was performed using canine-specific or cross-reactive antibodies against CD11b, CD14 and MHCII. In the blood of dogs with lymphoma (NRG and RG), compared to that of healthy ones, the MHCII+ and MHCII− monocytes ratio was changed due to an increase in the percentage of MHCII− monocytes. The number of MHCII− monocytes was significantly higher only in RG dogs compared to healthy ones, which might result from the release of these cells from the blood marginal pool due to the action of glucocorticoids. Our results encourage further studies to assess if changes in MHCII expression affect immune status in dogs with lymphoma.

Clinical and Immunological Features of SARS-CoV-2 Breakthrough Infections in Vaccinated Individuals Requiring Hospitalization

Background and PurposeWaning immunity and the surge of SARS-CoV-2 variants are responsible for breakthrough infections, i.e., infections in fully vaccinated individuals. Although the majority of vaccinated infected subjects report mild or no symptoms, some others require hospitalization. The clinical and immunological features of vaccinated hospitalized COVID-19 patients are currently unknown.MethodsTwenty-nine unvaccinated and 36 vaccinated hospitalized COVID-19 patients were prospectively enrolled and clinical and laboratory data were gathered. Immunophenotyping of leukocytes’ subsets, T and B cell SARS-CoV-2-specific responses were evaluated via flow cytometry. Anti-IFN-α autoantibodies were measured via ELISA.ResultsDespite vaccinated patients were older and with more comorbidities, unvaccinated subjects showed higher levels of pro-inflammatory markers, more severe disease, and increased mortality rate. Accordingly, they presented significant alterations in the circulating leukocyte composition, typical of severe COVID-19. Vaccinated patients displayed higher levels of anti-Spike IgGs and Spike-specific B cells. Of all participants, survivors showed higher levels of anti-Spike IgGs and Spike-specific CD4+ T cells than non-survivors. At hospital admission, 6 out of 65 patients (9.2%) displayed high serum concentrations of autoantibodies targeting IFN-α. Remarkably, 3 were unvaccinated and eventually died, while the other 3 were vaccinated and survived.ConclusionDespite more severe pre-existing clinical conditions, vaccinated patients have good outcome. A rapid activation of anti-SARS-CoV-2-specific immunity is fundamental for the resolution of the infection. Therefore, prior immunization through vaccination provides a significant contribution to prevention of disease worsening and can even overcome the presence of high-risk factors (i.e., older age, comorbidities, anti-IFN-α autoantibodies).

COVID-19 rapidly increases MDSCs and prolongs innate immune dysfunctions.

We used unsupervised immunophenotyping of blood leukocytes and measured cytokine production by innate immune cell exposed to LPS and R848. We show that COVID-19 induces a rapid, transient upregulation of myeloid-derived suppressor cells (MDSCs) accompanied by a rapid, sustained (up to 3 months) hyporesponsiveness of dendritic cells and monocytes. Blood MDSCs may represent biomarkers and targets for intervention strategies in COVID-19 patients.

PB1854: NEXT GENERATION SEQUENCING APPROACHES IN CHRONIC LYMPHOCYTIC LEUKAEMIA: FINDINGS FROM EXTERNAL QUALITY ASSESSMENT

Background: Chronic lymphocytic leukaemia (CLL) represents a clinically heterogeneous, lymphoproliferative neoplasm resulting in chronic B-lymphocyte proliferation. Historically, large cytogenetic aberrations detected by fluorescent in situ hybridisation were identifiable in patients, enabling prognostic assessment. With the development of next generation sequencing (NGS), variants have been identified in several genes that are recurrently mutated in CLL, including TP53, ATM, BIRC3, NOTCH1 and SF3B1, with the prognostic implications of variants in these genes being reported. However, only TP53 variant status has been incorporated into routine clinical diagnostics to guide prognosis, risk stratification and appropriate therapeutic interventions based on findings indicating decreased survival and resistance to immunochemotherapy. Aims: The aims of this study were to assess NGS gene panel content and design approaches in CLL to determine levels of harmonisation amongst laboratories. Methods: The United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping has provided external quality assessment for use of NGS technologies in CLL since 2018. The programme routinely requests NGS panel information (gene content and exon coverage) and other methodological data from participating laboratories. Data returns from 2018-2021 relating to NGS gene content and panel analysis were evaluated. Results: In total, 127 genes were analysed across 34 laboratories, ranging from 1-82 genes analysed in the context of CLL per laboratory (median of 12 genes). Of the 34 laboratories evaluated, five analysed TP53 only, with four of these utilising a non-disease specific myeloid NGS panel. The most commonly analysed genes were TP53 (100% laboratories), SF3B1 (73.5%) and NOTCH1 (70.6%). For these genes, there was a high-level of standardisation in sequencing approaches; with 91.2% of laboratories sequencing coding exons 2-11 in TP53 and 100% laboratories sequencing ‘hotspot’ exon 34 of NOTCH1, accounting for >80% variants in NOTCH1 associated with CLL. For SF3B1, 87% laboratories sequenced exons 14-16, which are hotspots for variants. Other frequently analysed genes included MYD88 (61.8% laboratories), BTK (58.8%), ATM and PLCG2 (52.9%). Summary/Conclusion: The number of genes analysed in the context of CLL amongst laboratories was highly variable. Use of NGS genetic testing approaches in haemato-oncology aids in accurate diagnosis and prognosis, however, the cost of developing these panels is not insubstantial and as such, laboratories often design panels to encompass multiple lymphoid/haematological neoplasms. Whilst recommendations outline the need for TP53 testing in CLL, the lack of technical and clinical guidelines on minimal NGS panel content and bioinformatic analysis that provide additional prognostic information has resulted in a wide variation in bioinformatic analysis approaches. This inevitably results in wide disparity of data generated and subsequent clinical interpretation. Guidelines are urgently needed for the identification of minimal NGS panel content in CLL and in other mature lymphoid neoplasms, outlining key genes directly involved in the diagnostic, prognostic and theranostic outputs in order to standardise patient testing and care.