Peripheral Blood Mononuclear Lymphocytes Research Articles

Comparative analysis of selected gene expression profiles in peripheral blood mononuclear cells in patients with rosacea and healthy volunteers

Introduction. Rosacea is a chronic skin disease with about 10% prevalence in the global population. The pathogenesis of the disease is multifactorial and is not yet fully understood. The dermatosis affects the facial skin and is characterized by persistent erythema, the formation of telangiectasias, papules, pustules, and can also be complicated by ophthalmic lesions.The disease is rooted in impaired neurovascular regulation and changes in the innate immune systems. It is known that the expression of vascular endothelial growth factors (VEGF) in the skin are increased in rosacea and the level of myeloperoxidase, a lymphocyte lysosomal enzyme, is increased in the inflammatory processes occurring in the arteries.Aim. To study the level of these gene expression in blood lymphocytes in the acute stage of the disease.Materials and methods. A total of 10 patients with erythematous rosacea, 10 patients with papulopustular rosacea and 10 healthy volunteers were included in the study. The peripheral blood samples were collected from the subjects for further isolation of lymphocytes. The level of gene expression was determined by real-time polymerase chain reaction.Results. Investigators observed a 3.7-fold increase in the activity of myeloperoxidase expression in immune blood cells compared to the values of healthy volunteers. Also, the level of relative VEGFA expression increased by 6.5 times, the level of VEGFС increased by 11 times in mononuclear lymphocytes of patients compared to the values of healthy subjects. A comparative analysis in the groups of patients with erythematous and papulopustular rosacea showed a multidirectional expression pattern of vascular growth factors VEGFA and VEGFС. An increase in VEGFA expression is observed in the papulopustular rosacea, while VEGFС is higher in the erythematous form of the pathology.Conclusion. The obtained results demonstrate that a significant increase in the gene expression levels associated with vascular activity and angiogenesis in rosacea occurs not only in the skin, but also in the peripheral blood mononuclear lymphocytes of the patients. The detected differences in the expression of vascular growth factors in different forms of the disease indicate the need for differentiated treatments.

SARS-CoV-2-Specific T Lymphocytes Analysis in mRNA-Vaccinated Patients with B-Cell Lymphoid Malignancies on Active Treatment.

Patients with B-lymphocyte malignancies (BCMs) receiving B-lymphocyte-targeted therapies have increased risk of severe COVID-19 outcomes and impaired antibody response to SARS-CoV-2 mRNA vaccination in comparison to non-hematologic oncologic patients or general population. Consequently, it is vital to explore vaccine-induced T-lymphocyte responses in patients referred for the understanding of immune protection against SARS-CoV2 infections. The objective of the present study was to analyze the recall immune responses carried out by T lymphocytes after two COVID-19 mRNA vaccine doses. We enrolled 40 patients with BCMs and 10 healthy controls (HCs) after 4 weeks from the second mRNA vaccine dose. Spike (S)-specific T-lymphocyte responses were assessed in peripheral blood mononuclear lymphocytes (PBMCs) by intracellular IFN-γ staining combined with flow cytometry. Furthermore, the humoral response was assessed with the measurement of anti-spike antibodies. From March to July 2021, 40 patients (median age 68) received mRNA vaccines. The overall antibody response for BCMs was 52.5% versus 100% for the healthy controls (p = 0.008). The antibody response was different across BCMs: 18.75% for non-Hodgkin lymphoma, 54.5% for chronic lymphocytic leukemia, and 92.3% for multiple myeloma. Responses varied by malignancy type and treatment, with anti-CD20 therapies showing the lowest response (6.7%). T-lymphocyte analysis revealed reduced numbers and altered differentiation stages in patients compared to the controls. However, the vaccine-induced T response was generally robust, with variations in specific T subpopulations. mRNA vaccines induced significant humoral and cellular immune responses in B-cell lymphoid malignancy patients, although responses varied by treatment type and malignancy. Further research is needed to optimize vaccination strategies in this population.

THE CO-ADMINISTRATION OF POIL-2 WITH TGEV INACTIVATED VACCINE ENHANCES IMMUNE RESPONSE OF PIGLETS TO TGEV

Porcine interleukin-2 (poIL-2) has not yet been demonstrated to be immune-enhancing against porcine transmissible gastroenteritis virus (TGEV) inactivated vaccine (IV), despite IL-2 having been proven to have immunological adjuvant effects for a variety of vaccinations. In this work, the impact of poIL-2 on TGEV IV in terms of immunological enhancement was investigated. Twenty four (24) SPF piglets were utilized and divided into six groups: PBS group, poIL-2 group, IV group, 10μg poIL-2+IV group, 50μg poIL-2 +IV group, and 250μg poIL-2+IV group. They received a second vaccine at 28 days point following the initial immunization. Serum and blood samples were obtained at various periods throughout the experiment. By using ELISA assay, neutralization assay, MTT assay, and flow cytometry assay, the TGEV-specific antibody expressions, neutralizing antibodies generations, interleukin-4 (IL-4), and interferon-gamma (IFN-γ) productions, peripheral blood mononuclear cells (PBMCs) proliferation response and lymphocyte phenotype subpopulations (CD3+, CD4+, and CD8+ immune cells) reflections were determined. The results showed that piglets inoculated with IV supplemented with poIL-2 significantly not only increased more piglet cellular immunity against TGEV by raising the degrees of IL-4, IFN-γ, Stimulation Index (SI), and the ratio of CD4+ /CD8+ cell subgroups, but also promoted more humoral immunity against TGEV by increasing levels of anti-TGEV specific antibodies and neutralizing antibodies (NAs) than those piglets inoculated with the TGEV IV alone. Additionally, the results suggested that porcine interleukin-2 (poIL-2) may improve pigs' immune responses in a dosage-dependent way.Our study revealed that poIL-2 had an immune-enhancing effect on the immunization of TGEV IV, and it possessed the potential to be applied as an immune-stimulating agent. Keywords: Inactivated vaccine (IV); adjuvant; porcine transmissible gastroenteritis virus (TGEV); porcine interleukin-2 (poIL-2); immune response.

Crohn's disease treatment and memory T-cell subset changes: insights from a case series.

Crohn's disease (CD) is a chronic inflammatory bowel disease with significant morbidity, affecting millions worldwide. The intricacies of immune responses in CD, especially post-treatment, remain a vital area of exploration. While memory T (Tm)-cell subsets play a pivotal role in adaptive immunity, their specific function in patients with CD after treatment is not well-understood. This study aims to investigate the effect and function of Tm-cell subsets in these patients, addressing a crucial knowledge gap in the context of CD therapeutics. A total of eight patients diagnosed with CD were selected based on predefined inclusion criteria. All patients were treated with either anti-inflammatory agents, immunosuppressive drugs, or a combination of both. For comparison, healthy donors were enrolled based on exclusion of autoimmune or inflammatory diseases. Peripheral blood mononuclear cells (PBMCs) and lymphocytes were isolated from blood and lymph node tissue respectively. The phenotype and cytokine production of T lymphocytes from both CD patients and healthy donors were analyzed using flow cytometry. Statistical comparisons of the outcomes between CD patients and healthy donors were made using Mann-Whitney test (two-tailed) and Student t-test. Post-treatment CD patients exhibited an altered T cell distribution with a notable increase in CD8+ T cells in PBMCs (P=0.0005), and altered frequencies of CD4+ and CD8+ T cells in mesenteric lymph nodes (MLNs). Tm cells showed decreased interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production, with significant alterations in the frequency of IFN-γ-producing CD8+ stem cell-like Tm (Tscm) cells in lesions of the MLNs from patients with CD (CD-M-Lys) compared to healthy MLNs from patients with CD (N-M-Lys) (P=0.0152). Differences in tissue-resident Tm (Trm)-cell subset frequencies were observed between the MLNs and small intestinal mucosa in CD patients. The treatments with anti-inflammatory agents and/or immunosuppressive drugs have a significant effect on the frequency and function of Tm-cell subsets. Clinically, these findings suggest a potential therapeutic avenue in modulating Tm-cell responses, which might be particularly beneficial for conditions where immune response modulation is crucial. Further clinical studies are warranted to explore the full therapeutic implications of these findings.

The Effect of Selegiline on Reducing Oxidative Stress in Polymorphonuclear Blood Cells (Lymphocytes) Isolated from Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a persistent condition resulting in an inflammatory response abide by the pathological mechanism of autoimmunity that causes the cartilage, bone, and joint tissues to deteriorate, lowering one’s quality of life in general. Several studies have suggested a significant association between oxidative stress caused by leukocyte-mediated inflammatory responses and the conversion of a substantial amount of oxygen into diverse free radicals, resulting in oxidative harm, including the chronic nature of rheumatoid arthritis. The utilization of DMARDs which are known as biologic and conventional disease-modifying antirheumatic drugs, has exhibited encouraging results. However, it is crucial to prioritize the discovery of novel and efficacious medications for rheumatoid arthritis in order to address the constraints associated with current therapeutic approaches. Selegiline, a synthetic drug primarily employed to manage Parkinson’s disease, predominantly interferes with the MAO-B enzyme, also known as monoamine oxidase. Additionally, it has been demonstrated to possess free radical-neutralizing properties. The goal of this study is to assess the effectiveness of selegiline in reducing oxidative stress indicators in peripheral blood mononuclear lymphocyte cells isolated from individuals diagnosed with RA. Peripheral blood was collected from 20 RA patients in accordance with the American College of Rheumatology standard. Lymphocytes were isolated following incubation with 1.5 μg/mL phorbol myristate acetate (PMA) and treatment with different concentrations (50–200 μg/mL) of selegiline. This study reveals that selegiline in concentrations of 150 and 200 μg/mL was effective enough to alleviate oxidative stress by scavenging free radicals and improving the level of natural defensive enzymes playing as antioxidants. Therefore, it can be concluded that the MAO-B inhibitor selegiline has potential as a non-toxic repurposed medicine for curtailing the pathological effects of oxidative overload during RA, unlocking the opportunity for further investigation into the impact on other inflammatory cells, such as neutrophils.

Feeding enhances fibronectin adherence of quiescent lymphocytes through non-canonical insulin signalling.

Nutritional availability during fasting and refeeding affects the temporal redistribution of lymphoid and myeloid immune cells among the circulating and tissue-resident pools. Conversely, nutritional imbalance and impaired glucose metabolism are associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent immune cell function and trafficking are scanty. Here, we report that oral glucose load in mice and healthy men enhances the adherence of circulating peripheral blood mononuclear cells (PBMCs) and lymphocytes to fibronectin. Adherence to fibronectin is also observed upon regular intake of breakfast following overnight fasting in healthy subjects. This glucose load-induced phenomenon is abrogated in streptozotocin-injected mice that lack insulin. Intra-vital microscopy in mice demonstrated that oral glucose feeding enhances the homing of PBMCs to injured blood vessels in vivo. Furthermore, employing flow cytometry, Western blotting and adhesion assays for PBMCs and Jurkat-T cells, we elucidate that insulin enhances fibronectin adherence of quiescent lymphocytes through non-canonical signalling involving insulin-like growth factor-1 receptor (IGF-1R) autophosphorylation, phospholipase C gamma-1 (PLCγ-1) Tyr783 phosphorylation and inside-out activation of β-integrins respectively. Our findings uncover the physiological relevance of post-prandial insulin spikes in regulating the adherence and trafficking of circulating quiescent T-cells through fibronectin-integrin interaction.

GLIS1 intervention enhances anti-PD1 therapy for hepatocellular carcinoma by targeting SGK1-STAT3-PD1 pathway

BackgroundGLI-similar 1 (GLIS1) is one of of Krüppel-like zinc finger proteins, which are either stimulators or inhibitors of genetic transcription. Nevertheless, its effects on T cell were elusive.MethodsIn this study,...

Depiction of immune heterogeneity of peripheral blood from patients with type II diabetic nephropathy based on mass cytometry.

Diabetic nephropathy (DN) is the most prominent cause of chronic kidney disease and end-stage renal failure. However, the pathophysiology of DN, especially the risk factors for early onset remains elusive. Increasing evidence has revealed the role of the innate immune system in developing DN, but relatively little is known about early immunological change that proceeds from overt DN. Herein, this work aims to investigate the immune-driven pathogenesis of DN using mass cytometry (CyTOF). The peripheral blood mononuclear lymphocytes (PBMC) from 6 patients with early-stage nephropathy and 7 type II diabetes patients without nephropathy were employed in the CyTOF test. A panel that contains 38 lineage markers was designed to monitor immune protein levels in PBMC. The unsupervised clustering analysis was performed to profile the proportion of individual cells. t-Distributed Stochastic Neighbor Embedding (t-SNE) was used to visualize the differences in DN patients' immune phenotypes. Comprehensive immune profiling revealed substantial immune system alterations in the early onset of DN, including the significant decline of B cells and the marked increase of monocytes. The level of CXCR3 was dramatically reduced in the different immune cellular subsets. The CyTOF data classified the fine-grained differential immune cell subsets in the early stage of DN. Innovatively, we identified several significant changed T cells, B cell, and monocyte subgroups in the early-stage DN associated with several potential biomarkers for developing DN, such as CTLA-4, CXCR3, PD-1, CD39, CCR4, and HLA-DR. Correlation analysis further demonstrated the robust relationship between above immune cell biomarkers and clinical parameters in the DN patients. Therefore, we provided a convincible view of understanding the immune-driven early pathogenesis of DN. Our findings exhibited that patients with DN are more susceptible to immune system disorders. The classification of fine-grained immune cell subsets in this present research might provide novel targets for the immunotherapy of DN.

Bioactive ZnO-assisted 1393 glass scaffold promotes osteogenic differentiation: Some studies.

We developed ZnO-assisted 1393 bioactive glass-based scaffold with suitable mechanical properties through foam replica technique and observed to be suitable for bone tissue engineering application. However, the developed scaffolds' ability to facilitate cellular infiltration and integration was further assessed through in vivo studies in suitable animal model. Herein, the pure 1393 bioactive glass (BG) and ZnO-assisted 1393 bioactive glass- (ZnBGs; 1, 2, 4mol% ZnO substitution for SiO2 in pure BG is named as Z1BG, Z2BG, Z3BG, respectively) based scaffolds were prepared through sol-gel route, followed by foam replica techniques and characterized by a series of in vitro and some in vivo tests. Different cell lines like normal mouse embryonic cells (NIH/3T3), mouse bone marrow stromal cells (mBMSc), peripheral blood mononuclear cells, that is, lymphocytes and monocytes (PBMC) and U2OS (carcinogenic human osteosarcoma cells) were used in determination and comparative analysis of the biological compatibility of the BG and ZnBGs. Also, the alkaline phosphatase (ALP) activity, and osteogenic gene expression by primer-specific osteopontin (OPN), osteocalcin (OCN), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were performed to study osteogenic differentiability of the stromal cells in different BGs. Moreover, radiological and histopathological tests were performed in bone defect model of Wister rats to evaluate the in vivo bone regeneration and healing. Interestingly, these studies demonstrate augmented biological compatibility, and superior osteogenic differentiation in ZnBGs, in particular Z3BG than the pure BG in most cases.

Changes in immunological parameters by ageing in rural healthy Indian adults and their associations with sex and lifestyle

Several factors including sex and lifestyle have been reported to contribute to the age-related alteration of immune functions. The study was undertaken to determine age-related differences in the proportion of peripheral blood mononuclear lymphocytes in the Indian population using blood samples from 67 healthy adults (33 females and 34 males) aged between 20 and 80 years old. In the linear regression analysis to estimate the relationship with age categories, there was a significant increase in the frequency of natural killer cells with ageing, while their cytolytic activity significantly declined. The frequency of CD4+ T cells increased with age, whereas that of CD8+ T cells decreased, resulting in the age-associated increase of the CD4/CD8 ratio. The subsets of B cells did not show any significant relationship with age. Although there were variations between the male and female subgroups in effect size of ageing, the trends were in the same direction in all the parameters. Reduced fat intake was associated with a lower frequency of CD4+ T cells, and higher serum cotinine level was associated with a higher CD4/CD8 ratio. The results indicate that cellular immunity in the Indian population is affected by ageing, while humoral immunity is less susceptible to ageing.

A sharp decrease of Th17, CXCR3+-Th17, and Th17.1 in peripheral blood is associated with an early anti-IL-17-mediated clinical remission in psoriasis.

Psoriasis—an immune-mediated skin disease—implicates in its pathophysiology by circulating pro-inflammatory cell populations, cytokines, and their interactions with the epidermis. The direct effect of approved anti-interleukin- (IL-)17A and anti-IL-17R biologic therapy on immunophenotyping of peripheral blood mononuclear lymphocytes’ (PBMCs) relative sub-population frequencies in psoriasis patients has not yet been described. Using multiparameter flow cytometry we examined T-cell subpopulations characterized by CCR6, CCR4, and CXCR3 chemokine receptor surface expression at baseline and after initiation of biologic therapy in PBMCs collected from 30 psoriasis patients. Increased CD3+CD4+CXCR3+, CD3+CD4+CCR6+CCR4+CXCR3+(CXCR3+-Th17), and CD3+CD4+CCR6+CCR4-CXCR3+(Th17.1) cell populations were observed in patients with psoriasis in comparison to healthy individuals (n = 10). IL-17 therapeutic blockade decreased CD3+CD4+CCR6+, CD3+CD4+CXCR3+, CD3+CD4+CCR6-CXCR3+(Th1), CD3+CD4+CCR6+CCR4+(Th17), CD3+CD4+CCR6+CCR4+CXCR3+(CXCR3+-Th17), and CD3+CD4+CCR6+CCR4-CXCR3+(Th17.1) cell populations in responding psoriasis patients. Moreover, CD3+CD4-CCR6+, CD3+CD4-CXCR3+, CD3+CD4-CCR6+CCR4+(Tc17), and CD3+CD4-CCR6-CXCR3+(Tc1) percentages were also inhibited. Modulation of the same cell sub-populations was also assessed in patients treated with methotrexate (n = 4), apremilast (n = 4), and anti-IL-23 biologic treatment (n = 4). In our study, the levels and functional capacity of peripheral pro-inflammatory Th1, Th17, and additional CCR6+T cell sub-gated populations from psoriasis patients that were treated with anti-IL-17 or anti-IL-17R targeted biologic therapy were explored for the first time. Our data clearly demonstrate that early anti-IL-17 mediated clinical remission is accompanied by a significant decrease of Th1, Th17, CXCR3+-Th17, and Th17.1 cells.

Circulating lymphocyte subsets are prognostic factors in patients with nasopharyngeal carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is a geographically and racially variable disease that has a high incidence in Southeast China. According to previous studies on tumor immunity, we compared multiple clinical parameters and blood indexes with outcomes regarding to Epstein-Barr virus (EBV) status in NPC patients.MethodsAccording to the EBV load at diagnosis, 220 NPC patients who received concurrent chemoradiotherapy (CRT) were divided into two groups: EBV DNA ≥ 1500 copies/mL and EBV DNA < 1500 copies/mL, respectively. We compared clinical parameters with peripheral blood mononuclear cells, lymphocyte subsets and biochemical indexes. We also analyzed distant metastases and the overall survival rate regarding to these characteristics.ResultsIn most cases, the two groups showed the same trends. Most blood indexes were decreased during CRT and the decrease of the absolute count was more significant than the percentage. Patients with younger age showed the higher CD3+ and CD3 + CD8+ percentages. Patients whose EBV DNA ≥ 1500 copies/mL showed a higher N classification than those with EBV DNA < 1500 copies/mL at first diagnosis. Within patients with EBV DNA ≥ 1500 copies/mL, a higher CD3 + CD8+ percentage or lower CD3-CD56+ percentage had better OS rates, and the CD3 + CD8+ percentage was an independent prognostic factor by multivariate survival analyses.ConclusionsCRT caused an overall decrease of blood cells in NPC patients. Among all the blood indexes, the CD3 + CD8+ percentage showed a correlation with age and was an independent prognostic factor in patients with EBV DNA ≥ 1500 copies/mL at first diagnosis, which is worthy for further large cohort study.

P341: A NOVEL BISPECIFIC T CELL ENGAGER (UMG2-CD3) IS EFFECTIVE AGAINST CORTICAL-DERIVED ACUTE LYMPHOBLASTIC LEUKEMIA

Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived by T cell precursors and characterized by poor prognosis.The immunotherapy has revolutionized the outcome of B cell acute lymphoblastic leukemia (B-ALL), but the absence of tumor-specific T cell antigen hampers its efficacy in T-ALL. Therefore, the development of novel immune-therapeutic options for the management of this orphan disease is eagerly awaited. CD1a is a glycoprotein expressed on cortical T-ALL and only on healthy thymocytes and Langerhans cells. Taking into account its safe pattern of expression, CD1a might represent a valuable therapeutic target for thetreatment of T-ALL. Aims: With the aim to provide an effective immune-therapeutic strategy for T-ALL, we developed a bispecific T cell engager (BTCE) derived from a novel UMG2 mAb that recognize a previously uncharacterized CD1a epitope. Methods: To evaluate the specificity of UMG2 binding to CD1a epitope, HEK293T cells which do not express CD1a endogenously, have been used. In this regards, cells were transfected with a plasmid that encode for CD1a or with an empty vector and UMG2 reactivity has been evaluated by flow cytometry. To assess the unicity of UMG2 mAb binding, a competitive binding assay between UMG2 and commercially available CD1a antibodies has been performed. The UMG2 expression profile on peripheral blood cells from healthy donors and on a panel of cortical T-ALL cells has been evaluated. To develop a UMG2-CD3 construct, an asymmetric 2 + 1 UMG2-CD3 bispecific T cellengager (BTCE) has been generated by using knobs into hole technology. UMG2-CD3 T cell-redirected cytotoxicity has been evaluated on HEK293T wild type, on HEK293T-CD1a+ and on patient-derived T-ALL cells, co-cultured with peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at different E:T ratio.Moreover, T cell activation has been assesed by flow cytometry. UMG2-CD3 anti-tumor activity against a CD1a+ T-ALL cells has been evaluated in vivo. For this purpose,Hu-PBMCs NSG mouse model has been generated and tumor growth has been assessed by fluorescent imaging probe via IVIS system. Results: UMG2 mAb recognizes a novel CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs with the exception of a partial competition with NA1/34-HLK clone. A strong UMG2 reactivity has been observed on T-ALL cells, while no binding has been found on normal blood cells. A concentration-dependent T cell cytotoxicity on CD1a+ T-ALL cells co-cultured with PBMCs in the presence of UMG2-CD3 has been observed. Minimal UMG2-CD3 residual anti-tumor activity has been observed in CD4/CD8 depleted and CD56 enriched lymphocytes. CD56 depleted and Fc-blocked BMCs were able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-CD3 could not recruit monocytes and NK cells through Fc-FcyR interaction. Moreover, the concentration-dependent increase of i) T cell proliferation, ii) cytotoxic degranulation marker (CD107a), iii) expression of cell surface activation markers (CD25, CD69),and iv) pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ) has been observed in the presence of UMG2-CD3. Importantly, in an in vivo immune-humanized NSG mice model,UMG2-CD3 was able to significantly inhibit tumor growth and then conferring the survival advantage of treated animals. Summary/Conclusion: All our findings, demonstrated that UMG2-CD3 BTCE represents a promising immune-therapeutic agent against T-ALL to be further investigated for clinical translation.

Abstract 1885: Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality

Abstract Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash࣪ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows. Citation Format: Melvin Lye, Christoph Eberle, Ann Wang, Geoffrey K. Feld, Namyong Kim. Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1885.

A novel aryl-guanidinium derivative, VP79s, targets the signal transducer and activator of transcription 3 signaling pathway, downregulates myeloid cell leukaemia-1 and exhibits preclinical activity against multiple myeloma

AimsWe have recently described a novel guanidinium-based compound, VP79s, which induces cytotoxicity in various cancer cell lines. Here, we aim to investigate the activity of VP79s and associated mechanisms of action in multiple myeloma (MM) cells in vitro and ex vivo. Main methodsThe effects of VP79s on cell viability and induction of apoptosis was examined in a panel of drug-sensitive and drug-resistant MM cell lines, as well as ex vivo patient samples and normal donor lymphocytes and platelets. Cell signaling pathways associated with the biological effects of VP79s were analysed by immunoblotting and flow cytometry. Gene expression changes were assessed by quantitative real-time PCR analysis. Key findingsVP79s was found to rapidly inhibit both constitutively active and IL-6-induced STAT3 signaling with concurrent downregulation of the IL-6 receptors, CD130 and CD126. VP79s induced a rapid and dose-dependent downregulation of anti-apoptotic Bcl-2 family member, myeloid cell leukaemia-1 (MCL-1). VP79s enhanced bortezomib induced cell death and was also found to overcome bone marrow stromal cell induced drug resistance. VP79s exhibited activity in ex vivo patient samples at concentrations which had no effect on peripheral blood mononuclear cells, lymphocytes and platelets isolated from healthy donors. SignificanceAs VP79s resulted in rapid inhibition of the key IL-6/STAT3 signaling pathway and downregulation of MCL-1 expression with subsequent selective anti-myeloma activity, VP79s may be a potential therapeutic agent with a novel mechanism of action in MM cells.

A Novel Bispecific T Cell Engager (UMG2-BTCE) Targeting CD1a-CD3ε Is Effective Against Cortical-Derived Acute Lymphoblastic Leukemia

Caterina Riillo*and Daniele Caracciolo*equally contributed to the work.Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and orphan hematological disease derived from malignant transformation of thymic T cell precursors. At present, the prognosis of relapsed/refractory patients remains poor. While immunotherapy has significantly improved the outcome of B cell acute lymphoblastic leukemia (B-ALL), the lack of tumor-restricted T cell antigens hampers its efficacy in T-ALL. (Caracciolo D, Riillo C, et al. Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia. J Immunother Cancer. 2021) Therefore, the development of novel immune-therapeutics is eagerly awaited.CD1a is a cell surface glycoprotein restricted to cortical T-ALL subtype expressed only by cortical thymocytes and Langerhans's cells among human healthy tissue and might represent a valuable therapeutic target for the treatment of T-ALL. On this basis, we develop an asymmetric monovalent 2 +1 bispecific T cell engager (BTCE) derived from a novel humanized UMG2 mAb directed against an original CD1a epitope selectively expressed by cortical T-ALL cells.Methods.To evaluate if UMG2 recognizes a specific CD1a epitope, HEK293T cell line, negative for CD1a expression, has been transfected with a plasmid encoding for CD1a (HEK293T/CD1a) and with a negative control vector and UMG2 reactivity has been evaluated by flow cytometry. A competitive binding assay between UMG2 and commercially available CD1a antibodies was performed. The UMG2 expression profile was evaluated on healthy donor peripheral blood cells and on a panel of cortical T-ALL cell lines. To develop a UMG2 targeting immunotherapeutic construct with a limited unspecific T cell activation, an asymmetric 2+1 UMG2-CD3 bispecific T cell engager (BTCE) was generated using knobs into holes technology. UMG2-CD3 BTCE in vitro T cell-mediated activity was evaluated on HEK293T CD1a antigen-negative cell line, on HEK293T/CD1a, on patient-derived and T-ALL cell lines, co-cultured with healthy donors derived peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at 10:1 E:T ratio.T cell activation, degranulation, proliferation, and pro-inflammatory cytokine secretion were assessed by flow cytometry on primary blasts and on T-ALL cells with effector lymphocytes.To evaluate UMG2-CD3 BTCE anti-tumor activity against CD1a expressing T-ALL cell line in vivo, Hu-PBMCs NSG mouse model was generated, and tumor growth was assessed by fluorescent imaging probe.ResultsUMG2 mAb recognizes a previously uncharacterized CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs. While a strong UMG2 reactivity is observed on both patient-derived samples and T-ALL cells, no binding is found on normal blood cells, indicating the tumor-restricted pattern of reactivity of UMG2.UMG2-CD3 BTCE specifically binds CD1a on leukemic cells and activates CD3ε downstream signaling pathway on T lymphocytes, as demonstrated by the concentration-dependent increase of T cell proliferation, cytotoxic degranulation (CD107a), expression of cell surface activation markers (CD25, CD69), and pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ).UMG2-CD3 BTCE mediates strong and concentration-dependent specific T cell re-directed cytotoxicity only on CD1a expressing leukemic cells in the presence of T lymphocytes. Minimal UMG2-CD3 BTCE residual anti-tumor activity is observed in CD4/CD8 depleted and CD56 enriched lymphocytes, while CD56 depleted and Fc-blocked PBMCs are able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-BTCE could not recruit monocytes and NK cells through Fc-FcyR interaction by reducing the risk of immune-mediated adverse events. Most importantly, in an in vivo of immune-humanized NSG mice engrafted with human T-ALL cells, UMG2-BTCE significantly inhibits tumor growth translating into the survival advantage of treated animals.Conclusion: Taken together, all these results provide a framework for the clinical development of UMG2-CD3 BTCE potentially offering a novel therapeutic path for cortical-derived T-ALL. DisclosuresNo relevant conflicts of interest to declare.

A pilot trial of vaccination with Carcinoembryonic antigen and Her2/neu peptides in advanced colorectal cancer.

Checkpoint-blockade therapy (CBT) is approved for select colorectal cancer (CRC) patents, but additional immunotherapeutic options are needed. We hypothesized that vaccination with carcinoembryonic antigen (CEA) and Her2/neu (Her2) peptides would be immunogenic and well tolerated by participants with advanced CRC. A pilot clinical trial (NCT00091286) was conducted in HLA-A2+ or -A3+ Stage IIIC-IV CRC patients. Participants were vaccinated weekly with CEA and Her2 peptides plus tetanus peptide and GM-CSF emulsified in Montanide ISA-51 adjuvant for 3 weeks. Adverse events (AEs) were recorded per NIH Common Terminology Criteria for Adverse Events version 3. Immunogenicity was evaluated by interferon-gamma ELISpot assay of in vitro sensitized peripheral blood mononuclear cells and lymphocytes from the sentinel immunized node. Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All 11 participants were included in AEs and survival analyses, and the 10 eligible participants were evaluated for immunogenicity. All participants reported AEs: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AEs; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival (OS) was 16 months; among those enrolled with no evidence of disease (n=3), median OS was not reached after 10 years of follow-up. These data demonstrate that vaccination with CEA or Her2 peptides is well tolerated and immunogenic. Further study is warranted to assess potential clinical benefits of vaccination in advanced CRC either alone or in combination with CBT.

HeberNasvac, a Therapeutic Vaccine for Chronic Hepatitis B, Stimulates Local and Systemic Markers of Innate Immunity: Potential Use in SARS-CoV-2 Postexposure Prophylaxis.

IntroductionMore than 180 million people have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and more than 4 million coronavirus disease-2019 (COVID-19) patients have died in 1.5 years of the pandemic. A novel therapeutic vaccine (NASVAC) has shown to be safe and to have immunomodulating and antiviral properties against chronic hepatitis B (CHB).Materials and methodsA phase I/II, open-label controlled and randomized clinical trial of NASVAC as a postexposure prophylaxis treatment was designed with the primary aim of assessing the local and systemic immunomodulatory effect of NASVAC in a cohort of suspected and SARS-CoV-2 risk-contact patients. A total of 46 patients, of both sexes, 60 years or older, presenting with symptoms of COVID-19 were enrolled in the study. Patients received NASVAC (100 μg per Ag per dose) via intranasal at days 1, 7, and 14 and sublingual, daily for 14 days.Results and discussionThe present study detected an increased expression of toll-like receptors (TLR)-related genes in nasopharyngeal tonsils, a relevant property considering these are surrogate markers of SARS protection in the mice model of lethal infection. The HLA-class II increased their expression in peripheral blood mononuclear cell's (PBMC's) monocytes and lymphocytes, which is an attractive property taking into account the functional impairment of innate immune cells from the periphery of COVID-19-infected subjects. NASVAC was safe and well tolerated by the patients with acute respiratory infections and evidenced a preliminary reduction in the number of days with symptoms that needs to be confirmed in larger studies.ConclusionsOur data justify the use of NASVAC as preemptive therapy or pre-/postexposure prophylaxis of SARS-CoV-2 and acute respiratory infections in general. The use of NASVAC or their active principles has potential as immunomodulatory prophylactic therapies in other antiviral settings like dengue as well as in malignancies like hepatocellular carcinoma where these markers have shown relation to disease progression.How to cite this articleFleites YA, Aguiar J, Cinza Z, et al. HeberNasvac, a Therapeutic Vaccine for Chronic Hepatitis B, Stimulates Local and Systemic Markers of Innate Immunity: Potential Use in SARS-CoV-2 Postexposure Prophylaxis. Euroasian J Hepato-Gastroenterol 2021;11(2):59–70.

A chitosan-based nanosystem as pneumococcal vaccine delivery platform.

Chitosan-based nanosystems have been described as interesting tools for antigen delivery and for enhancing the immunogenicity of nasally administered vaccines. As a possible vaccine delivery method, the chemical conjugation of chitosan nanocapsules with the Streptococcus pneumoniae cell membrane protein PsaA (pneumococcal surface adhesin A) is suggested here. The antigen PsaA, common to all pneumococcus serotypes, is expected to improve its uptake by immune cells and to activate specific T cells, generating an adaptive immune response against pneumococcus.With this aim, chitosan nanocapsules with thiol-maleimide conjugation between the polymer (chitosan) and the antigen (PsaA) were designed to enable the surface presentation of PsaA for immune cell recognition. Spherical-shaped particles, with a size of 266 ± 32nm, positive charge of +30 ± 1mV, and good stability profiles in simulated nasal fluids (up to 24h) were achieved. PsaA association rates were three times higher compared with nanocapsules without covalent polymer-protein conjugation. Cytotoxicity studies in cell culture media showed non-toxic effect under 150µg/mL concentration of nanocapsules, and subsequent studies on the maturation of immature dendritic cells in the presence of antigen-conjugated nanocapsules displayed peripheral blood mononuclear cell activation and lymphocyte differentiation after their presentation by dendritic cells. Secretion of TNFα following exposure to nanocapsules and the ability of nanocapsules to activate CD4 and CD8 T lymphocytes had also been studied. Antigen loaded nanocarrier uptake and presentation by professional presenting cells.

Effects of IL-27 on Th17 Cells in Patients with Henoch-Schönlein Purpura

To investigate the effect of IL-27 on Th17 cells in patients with henoch-schönlein purpura（HSP） in order to further elucidate the pathogenesis. Fifty patients with HSP treated in our hospital from April 2019 to July 2019 were selected as HSP group, and 30 volunteers underwent physical examination at the same time were selected as control group. The proportion of Th17 cells in peripheral blood of HSP group and healthy control group was determined by flow cytometry (FCM). A total of 27 HSP patients were selected, and candidate peripheral blood mononuclear lymphocytes (PBMC) were co-cultured with exogenous rhIL-27, and the ratio of Th17 cells was detected by flow cytometry. The proportion of Th17 cells in the peripheral blood of HSP patients with acute phase was (1.57±0.54)%, which was significantly higher than that of the control group (0.86±0.40)% (t=-6.298, P<0.001), and the proportion of Th17 cells was decreased significantly after rhIL-27 co-culture (1.39%±0.52% vs 0.98%±0.44%)(P<0.05). IL-27 can reduce the level of Th17 cells in patients with HSP, which may be involved in the pathogenic process of HSP and play a protective role in the development of the disease.