CD4 Molecule Research Articles

Novel Peptide-Based Plectin-1 CAR T-Cells Target Specifically Pancreatic Cancer Cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating cancers, and therapies to treat this malignancy have not evolved rapidly to fulfill the need of affected patients. PDAC, like many solid malignancies, is considered a “cold immunological tumor” where the lack of targetable tumor-associated antigens has hampered the development of successful immunotherapies. Despite newer treatment approaches, PDAC patients with advanced disease have a dismal prognosis. Therefore, there is an urgent need to develop new therapies. Plectin-1 is a high molecular weight protein (∼500 kDa) that participates in cytoskeleton network organization linking intermediate filaments to microtubules and microfilaments. Plectin-1 is aberrantly expressed in the PDAC cellular membrane, contributes to PDAC pathogenesis, and confers a poor prognosis. Plectin-1-targeting peptides (PTP) in mice and humans have been successfully explored for their use in PDAC imaging and targeted payload delivery (small molecules, radioisotopes, and chemotherapy). We sought to take advantage of the specific binding of PTP and develop a peptide-based plectin-1 chimeric antigen receptor (CAR), and test T-cell transduced with this construct in vitro against PDAC cell lines. Methods: Using in silico tools, we optimized different pepto-CAR constructs and selected one that showed the highest levels of cytotoxicity against plectin-1-expressing PDAC cell lines. A myc tag was incorporated in the hinge region for flow cytometry or western blot detection. The intracellular signaling domain of these CAR sequences included 4-1BB and the zeta chain of the CD3 molecule ( Figure 1A). Plasmids containing the CAR sequence were packaged on lentivirus particles using HEK293T cells. Purified plectin-1 CAR lentiviruses were used to transduce T-cells derived from human peripheral blood T-cells obtained and purified from healthy donor whole blood. The expanded CAR T-cells were used for cytotoxic experiments against different cell lines. Results: Expression of plectin-1 CAR was confirmed by flow cytometry using an anti-myc tag antibody ( Figure 1B). Plectin-1 expression was analyzed in different pancreatic cancer cell lines, and the BxPC-3 cells showed the highest expression level ( Figure 1B). Using a luciferase-based assay, we plated 50,000 BxPC-3 cells per well in duplicates with different effector-to-target (E:T) ratios. After 24 hours, luciferin was added, and the plate was read using a luminometer. As a negative control for effector cells, we use T-cells without a CAR ( Figure 1B), and for the target cells, we use the plectin-1 negative lung cancer cell line A549 ( Figure 1B). Plectin-1 CAR T-cells effectively killed BxPC-3 cells at different E:T ratios in a specific manner compared with controls using unmodified T-cells. Furthermore, no cytotoxicity was observed against plectin-1 negative A549 cells ( Figure 1A). Conclusion: Our results show that a novel peptide-based CAR construct design against plectin-1 can induce specific cytotoxicity on plectin-1 expressing PDAC cell line BxPC-3. Furthermore, these data demonstrate the feasibility of using a CAR with a peptide-based antigen binding domain instead of the traditional antibody-derived scFv. This novel peptide-based CAR design offers alternatives for cellular therapy bioengineering. Moreover, our results can significantly impact immune-oncology therapeutic applications, particularly in challenging diseases like pancreatic cancer. Figure 1 . A) In the above section, the Plectin-1 CAR construct is depicted. Cytotoxicity of unmodified T-cells and Plectin-1 CAR T-cells against BxPC-3 and A549 is shown below. B) Expression of Plectin-1 CAR in unmodified T-cells and Plectin-1 CAR T-cells is shown on the left. The expression of plectin-1 antigen on the surface of BxPC-3 cells and A549 cells is shown on the right. PTP: plectin-1 targeting peptide.

Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE

AbstractImmunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G–Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

Identification of Potential Sex-Specific Biomarkers in Pigs with Low and High Intramuscular Fat Content Using Integrated Bioinformatics and Machine Learning.

Intramuscular fat (IMF) content is a key determinant of pork quality. Controlling the genetic and physiological factors of IMF and the expression patterns of various genes is important for regulating the IMF content and improving meat quality in pig breeding. Growing evidence has suggested the role of genetic factors and breeds in IMF deposition; however, research on the sex factors of IMF deposition is still lacking. The present study aimed to identify potential sex-specific biomarkers strongly associated with IMF deposition in low- and high-IMF pig populations. The GSE144780 expression dataset of IMF deposition-related genes were obtained from the Gene Expression Omnibus. Initially, differentially expressed genes (DEGs) were detected in male and female low-IMF (162 DEGs, including 64 up- and 98 down-regulated genes) and high-IMF pigs (202 DEGs, including 147 up- and 55 down-regulated genes). Moreover, hub genes were screened via PPI network construction. Furthermore, hub genes were screened for potential sex-specific biomarkers using the least absolute shrinkage and selection operator machine learning algorithm, and sex-specific biomarkers in low-IMF (troponin I (TNNI1), myosin light chain 9(MYL9), and serpin family C member 1(SERPINC1)) and high-IMF pigs (CD4 molecule (CD4), CD2 molecule (CD2), and amine oxidase copper-containing 2(AOC2)) were identified, and then verified by quantitative real-time PCR (qRT-PCR) in semimembranosus muscles. Additionally, the gene set enrichment analysis and single-sample gene set enrichment analysis of hallmark gene sets were collectively performed on the identified biomarkers. Finally, the transcription factor-biomarker and lncRNA-miRNA-mRNA (biomarker) networks were predicted. The identified potential sex-specific biomarkers may provide new insights into the molecular mechanisms of IMF deposition and the beneficial foundation for improving meat quality in pig breeding.

The HLA class I immunopeptidomes of AAV capsid proteins.

Cellular immune responses against AAV vector capsid represent an obstacle for successful gene therapy. Previous studies have used overlapping peptides spanning the entire capsid sequence to identify T cell epitopes recognized by AAV-specific CD8+ T cells. However, the repertoire of peptides naturally displayed by HLA class I molecules for CD8 T cell recognition is unknown. Using mRNA transfected monocyte-derived dendritic cells (MDDCs) and MHC-associated peptide proteomics (MAPPs), we identified the HLA class I immunopeptidomes of AAV2, AAV6 and AAV9 capsids. MDDCs were isolated from a panel of healthy donors that have diverse alleles across the US population. mRNA-transfected MDDCs were lysed, the peptide:HLA complexes immunoprecipitated, and peptides eluted and analyzed by mass spectrometry. We identified 65 AAV capsid-derived peptides loaded on HLA class I molecules of mRNA transfected monocyte derived dendritic cells. The HLA class I peptides are distributed along the entire capsid and more than 60% are contained within HLA class II clusters. Most of the peptides are organized as single species, however we identified twelve clusters containing at least 2 peptides of different lengths. Only 9% of the identified peptides have been previously identified as T cell epitopes, demonstrating that the immunogenicity potential for the vast majority of the AAV HLA class I immunopeptidome remains uncharacterized. In contrast, 12 immunogenic epitopes identified before were not found to be naturally processed in our study. Remarkably, 11 naturally presented AAV peptides were highly conserved among the three serotypes analyzed suggesting the possibility of cross-reactive AAV-specific CD8 T cells. This work is the first comprehensive study identifying the naturally displayed HLA class I peptides derived from the capsid of AAVs. The results from this study can be used to generate strategies to assess immunogenicity risk and cross-reactivity among serotypes during gene therapies.

Microglial expression of CD83 governs cellular activation and restrains neuroinflammation in experimental autoimmune encephalomyelitis

Microglial activation during neuroinflammation is crucial for coordinating the immune response against neuronal tissue, and the initial response of microglia determines the severity of neuro-inflammatory diseases. The CD83 molecule has been recently shown to modulate the activation status of dendritic cells and macrophages. Although the expression of CD83 is associated with early microglia activation in various disease settings, its functional relevance for microglial biology has been elusive. Here, we describe a thorough assessment of CD83 regulation in microglia and show that CD83 expression in murine microglia is not only associated with cellular activation but also with pro-resolving functions. Using single-cell RNA-sequencing, we reveal that conditional deletion of CD83 results in an over-activated state during neuroinflammation in the experimental autoimmune encephalomyelitis model. Subsequently, CD83-deficient microglia recruit more pathogenic immune cells to the central nervous system, deteriorating resolving mechanisms and exacerbating the disease. Thus, CD83 in murine microglia orchestrates cellular activation and, consequently, also the resolution of neuroinflammation.

SARS-CoV-2 uses CD4 to infect T helper lymphocytes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Construction of CD19 targeted dual- and enhanced dual-antibodies and their efficiency in the treatment of B cell malignancy

BackgroundT cell-redirecting bispecific antibodies establish a connection between endogenous T cells and tumor cells, activating T cells function to eliminate tumor cells without ex vivo genetic alteration or manipulation. Here, we developed a novel dual-specific antibody (DuAb) and an enhanced DuAb (EDuAb) with different stimulation signal to activate T cells, and evaluated their impact on the treatment of acute lymphoblastic leukemia (ALL).MethodsThe expression plasmids of the DuAb and EDuAb containing CD80 molecule were constructed by cloning heavy chain and light chain variable fragments from anti-human CD19 (HI19a) and CD3 (HIT3a) monoclonal antibody hybridomas, respectively. The activation and the anti-tumor efficacy of human T cells mediated by DuAb and EDuAb were evaluated in vitro. B-cell ALL xenograft NSG mouse model was established to investigate the therapeutic effect in vivo.ResultsEDuAb promoted the optimal expansion of primary human T cells with low expression of inhibitory markers in vitro than DuAb did. Both DuAb and EDuAb showed a similar capability in inducing healthy donor T cells to specifically eliminate B-ALL cell lines and primary blasts from patients. The similar ability was also observed in the patient-derived T cells. In vivo study showed that both DuAb and EDuAb significantly alleviated tumor burden and extended survival of B-ALL xenograft NSG mice. The median survival of PBS, DuAb and EDuAb treatment groups were 27, 38 and 45 days, respectively. The phenotype of T cells and cytokine release in peripheral blood (PB) of B-ALL xenograft NSG mice on day 24 were analyzed as well. The results showed that the proportion of CD8+ T cells and cytokine levels, including IL-2, IFN-γ and TNF-α, were higher in the EDuAb group than that of DuAb. Moreover, both DuAb and EDuAb significantly decreased the residual leukemia cells in PB of B-ALL xenograft NSG mice.ConclusionsBoth DuAb and EDuAb showed great potential as novel treatments for B-ALL in clinical applications. However, compared to DuAb, EDuAb showed a significant advantage in promoting the proliferation and survival of T cells. Furthermore, EDuAb showed a better promising effect on eliminating tumor cells and extending survival in vivo, which provides new insights for the development of new multi-specific antibodies.

202-OR: Automated Cluster Analysis by Machine Learning Reveals Important Dissimilarities in the Immune Phenotype of T1D, MS, and SLE

Background: Dysfunction of the immune cell control and break-down of self-tolerance are important in chronic conditions such as type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Investigating common patterns and dissimilarities between these diseases can help identifying new therapeutic intervention strategies for T1D. Methods: Unsupervised, automated gating and machine learning (ML) was used to identify clusters of immune cell phenotypes in fluorochrome stained PBMCs from patients with T1D (n=70), RA (n=70), SLE (n=50), MS (n=60) and healthy controls (n=70). The pipeline includes unsupervised pre-gating, FlowSOM clustering, and a statistical analysis (MANOVA) to check for significant differential abundances of cell populations among the conditions. Results: The unsupervised analysis revealed important differences in the immune cell phenotype of peripheral CD4posT cells between the different auto-immune conditions. IL7alpha receptor (CD127) positive T cell clusters, such as CD3hiCD4hiCD15sposCD127hiCD161med cells are significantly decreased in T1D (p<0.001) compared to MS. Clusters of IL7Rpos memory T cells, expressing the co-stimulatory CD28 molecule are significantly increased in T1D (p<0.001) and SLE (p<0.001) compared to MS. Clusters including Treg-like phenotypes are significantly decreased in MS (p<0.001) compared to T1D, SLE and RA. Discussion: IL-7R (CD127) and its gene polymorphism plays a major role in MS and mediates homeostatic proliferation in T1D. In SLE serum sIL-7R is associated with disease activity. Using unsupervised machine learning we were able to identify interesting clusters of immune cells expressing CD127 that show significantly different abundancies. Learning from immune modifying therapies used in MS and SLE treatment could help to develop new strategies for future T1D (combinational)-therapies targeting IL7/IL7R complexes. Disclosure B.Prietl: None. J.Vera-ramos: None. V.Pfeifer: None. M.Khalil: Advisory Panel; Novartis, Biogen, Gilead Sciences, Inc., Merck & Co., Inc., Research Support; Novartis, Biogen, Roche Pharmaceuticals. L.Herbsthofer: None. H.Sourij: Advisory Panel; Amgen Inc., Amryt Pharma Plc, Boehringer Ingelheim Inc., Eli Lilly and Company, Novo Nordisk A/S, Sanofi, Speaker's Bureau; Boehringer Ingelheim Inc., Novo Nordisk A/S, Bayer Inc., Sanofi. T.Pieber: Advisory Panel; Arecor, Novo Nordisk A/S, Consultant; Lilly, Research Support; AstraZeneca, Novo Nordisk A/S, Sanofi, Speaker's Bureau; Roche Diagnostics. Funding JDRF/Lupus Research Alliance/National MS Society Joint RFA (2-SRA-2021-1043-S-B)

Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4+ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4+ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4+ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.

#5337 PHENOTYPIC CHANGES OF LYMPHOCYTES FOLLOWING RENAL TRANSPLANTATION

Abstract Background and Aims Phenotypic changes in lymphocytes have been described following renal transplantation (RT), including both CD4 and CD8 subpopulations. The present work aims to prospectively assess of T lymphocyte phenotypic alterations, after RT and compare differences between deceased (DR) and living (LR) donor recipients. Method One hundred six RTRs were included in the study, 78/106 (74%) DR, age 52.5(15)yrs and 28/106 (26%) LD, 35(21)yrs. Lymphocytes, CD4, CD8, CD4CD28null, CD8CD28null, CD16+CD56+ (NK) and CD4+CD25+FoXP3+ (Tregs) were evaluated at peripheral blood, by flow cytometry, at certain time points: RT and 3, 6, 12 months (T0, T3, T6, T12, respectively). Results During follow up, at T3, T6, T12, eGFR was 61(23.6), 62.2(31.1), 63.5(23.9)ml/min/1.73 m2, p=NS and 59.1(24.8), 61(25.3), 62(22.7), p=NS, in DR and LR group, respectively. eGFR showed no significant difference between DR and LR at any time point. At any time point, T0, T3, T6, 12, lymphocytes were significantly increased in DR compared to LR groups, P = .001, p<0.0001, P = .002, p<0.0001, similarly CD4, P = .001, p<0.0001, P = .001, p<0.0001, and CD8 cells, P = .002, p<0.0001, P = .005, P = .006, respectively. Tregs were increased at T0, T3, T6, not at T12, p<0.0001, p<0.0001, P = .001, P = .1, respectively. At this point, Tregs were increased in RTRs with eGFR>50ml/min/1.73 m2, 20.3(9.3) vs. 27.2(18.6), P = .03. CD4CD28null, CD8CD28null cells did not change during follow up, and there was no difference between two groups at any time point. Conclusion Immune profile, was improved in both groups, DR or LR, however a significantly better effect managed in LR, although elimination of CD28 molecule could not be restored. Interestingly, Tregs were associated only by renal function one year following RT.

Antigen presenting subset of СD66b<sup>+</sup>CD16<sup>+</sup>CD33<sup>+</sup>HLA-DR<sup>+</sup> neutrophilic granulocytes in acute osteomyelitis in children: Immunomodulating effects of immunotropic hexapeptide in an <i>in vitro</i> experimental system

Inclusion of neutrophilic granulocytes (NG) in inflammation depends on the expression of receptors providing the functions of NG. Acute osteomyelitis (AOM) occupies a central place among purulentinflammatory diseases in children. AOM purulent-necrotic process proceeds in the bone, bone marrow – the site of hematopoiesis. It is interesting to determine the functionally significant NG subsets, their phenotype in OM and evaluate the effect of immunotropic substances for the correction of dysfunctions. Aim: to specify the variants of changes in quantitative and phenotypic characteristics of CD66b+CD16+CD33+HLA-DR-, CD66b+CD16+CD33+HLA-DR+ NG subsets at AOM in children and evaluate the possibility of their immunomodulation under the influence of hexapeptide (HP) – Arginyl-alpha-Aspartyl-Lysyl-Valyl-Tyrosyl-Arginine in vitro.Peripheral blood (PB) of 24 children 8-15 years old AOM were the study group (SG). The comparison group (CG) – 13 healthy children. HP (10-6 g/L) were incubated with PB SG (60 min, 37 °C) to evaluate the effects (SG1). The number of NG subsets CD66b+CD16+CD33+HLA-DR+, CD66b+CD16+CD33+HLA-DR- (FC500, Beckman Coulter, USA), receptor expression density (MFI), phagocytic activity before and after incubation with HP were determined.The NG subset expressing HLA-DR – 29.9 (18.4-43.6) % CD66b+CD16+CD33+HLA-DR+ was registered in children with AOM. The number of CD66b+CD16+CD33+HLA-DR+ was 1.5 times lower (p > 0.05), of CD66b+CD16+CD33+HLA-DR+ was 1.2 times higher (p > 0.05) than before incubation with of HP. The redistribution of subsets apparently occurs due to the binding of HPs to HLA-DR on the NG membrane. Also MFI HLA-DR was low (p > 0.05); the 1.3-fold increase in MFI CD66b, 1.4-fold decrease in MFI CD16 were revealed (p < 0.05).The study was the first to demonstrate the presence of NG subset of CD66b+CD16+CD33+HLA-DR+ in the PB of children with AOM. Subset of CD66b+CD16+CD33+HLA-DR+NG in AOM indicates the appearance of an activated subset of NG in PB with the properties of APC. The positive influence of HP on the phenotypic characteristics of subsets СD66b+CD16+CD33+HLA-DR-, СD66b+CD16+CD33+HLA-DR+. Restoration of phagocytic function of NGs under the influence of HP is connected with the increase of CD66b expression, which influences the effector function of NGs and decrease of CD16 molecule hyperexpression that stipulates decrease of damaging cytotoxic activity of NGs.

Transcriptomic analysis of regenerated skins reveals key genes involved in skin immune response and air-breathing of loach (Paramisgurnus dabryanus)

Fish skin acts as the first immune barrier against pathogens from external environments. Recently, fish skin immunity research has gradually become a hot topic in aquaculture disease control. Interestingly, the skin can do air-breathing in some bimodal respiration fish. However, there is no comprehensive understanding of these two functions. In this study, the skin regeneration of loach (Paramisgurnus dabryanus) was investigated through morphological and histological observations. Then, original skins (OS) and the regenerated skins (RS) when their capillaries were the most abundant during healing, were collected for transcriptomic analysis. 285,899,386 clean reads with a total length of 42.34 Gb were obtained. A total of 1282 differentially expressed genes (DEGs) were detected, including 1030 DEGs up-regulated and 252 DEGs down-regulated in the comparison of RS vs. OS. Fc gamma R-mediated phagocytosis (containing gelsolin (Gsn)), chemokine signaling pathway (containing C–C motif chemokine ligand 28 (Ccl28)), and B cell receptor signaling pathway (containing CD80 molecule (Cd81)) were closely related to skin immune response of the loach. PI3K-Akt signaling pathway [containing fibronectin 1 (Fn1) and lysyl oxidase-like 2b (Loxl2b)], TGF-beta signaling pathway [containing thrombospondin 1 (Thbs1)], ECM-receptor interaction [containing integrin alpha 7 (Itga7), Itgb8, Itgb10 and Itgb5], and dilated cardiomyopathy [containing laminin subunit alpha1 (Lama1)] were closely associated with skin air-breathing of the loach. This study is conducive to explore the molecular mechanisms of skin immune response during its regeneration and air-breathing of bimodal respiration fish. This study will benefit for the aquaculture of P. dabryanus and its related species.

Principle and Progress of Treating Lymphoma with CD22 as Immunotherapy Target

B-cell lymphoma is a type of lymphoid tissue tumour, which is usually caused by clonal proliferation of lymphocytes at different stages of differentiation. CD22 molecule is ubiquitous in normal B cells and B cell malignant tumours. It is a principal element in the B cell maturation and proliferation, and provides an important functional link to the adjustment of B cell surface and intracellular signaling and cell survival. Although the function of CD22 remains unclear, blocking the action of CD22 has been proved to inhibit the growth of malignant B cells and CD22 has become an important therapeutic target of B-cell lymphoma. We will also discuss the strategies for the treatment of B-cell lymphoma targeting CD22, including naked antibodies, bispecific antibodies, antibody-drug conjugate (ADC), antibodies conjugated to radioactive elements, and chimeric antigen receptor-based T-cell (CAR-T) cell immunotherapy.

Effect of Epstein-Barr Virus Infection on Selected Immunological Parameters in Children with Type 1 Diabetes.

Diabetes mellitus is a group of metabolic disorders with different etiologies, pathogeneses and clinical pictures, characterized by chronic hyperglycemia due to abnormal insulin secretion or action. Type 1 diabetes mellitus is the most common type of diabetes mellitus in children and adolescents, accounting for about 90% of diabetes in the population under the age of 18. The etiopathogenesis of type 1 diabetes is multifactorial. The disease occurs as a result of the interaction of three factors: genetic predisposition, environmental factors and the immune response. Research in recent years has focused on the involvement of Epstein-Barr virus (EBV) in the pathogenesis of type I diabetes. The goals of treating type 1 diabetes include maintaining blood-glucose, fructosamine and glycated hemoglobin (HbA1c) levels; therefore, the main purpose of this study was to evaluate the effect of EBV infection on the activation of selected immune cells, fructosamine levels and HbA1c levels in children with type I diabetes. Based on our study, we found a lower percentage of CD8+ T lymphocytes with expression of the CD69 molecule in patients with anti-VCA antibodies in the IgG class, and a lower percentage of CD8+ T lymphocytes with expression of the CD25+ molecule in patients with anti-EBNA-1 antibodies in the IgG class, which may indicate limited control of the immune system during EBV infection in patients. There was a lower percentage of CD3+CD4+ T lymphocytes secreting IL-4 in the study group, indicating that a deficiency in IL-4 production may be related to the development of type 1 diabetes. There was an increase in the percentage of CD4+CD3+IL-10 lymphocytes in the study group with anti-VCA antibodies present in the IgG class and anti-EBNA-1 antibodies in the IgG class compared to the patients without antibodies. In addition, there was a significant increase in fructosamine levels and higher glycated hemoglobin levels in the study group with antibodies to EBV antigens. In addition, an increase in the percentage of T lymphocytes with a CD4+CD3+IL-17+ phenotype in the patients with anti-VCA IgG antibodies was confirmed, and higher HbA1c levels may suggest that EBV infection is accompanied by an increase in IL-17 secretion.

CD83 acts as immediate early response gene in activated macrophages and exhibits specific intracellular trafficking properties

Macrophages (MΦ) are remarkably plastic cells, which assume phenotypes in every shade between a pro-inflammatory classical activation, and anti-inflammatory or resolving activation. Therefore, elucidation of mechanisms involved in shaping MΦ plasticity and function is key to understand their role during immunological balance. The immune-modulating CD83 molecule is expressed on activated immune cells and various tissue resident MΦ, rendering it an interesting candidate for affecting MΦ biology. However, in-depth analyses of the precise kinetics and trafficking of CD83 within pro-inflammatory, LPS activated bone-marrow-derived MΦ have not been performed. In this study, we show that activation with LPS leads to a very fast and strong, but transient increase of CD83 expression on these cells. Its expression peaks within 2 h of stimulation and is thereby faster than the early activation antigen CD69. To trace the CD83 trafficking through MΦs, we employed multiple inhibitors, thereby revealing a de novo synthesis and transport of the protein to the cell surface followed by lysosomal degradation, all within 6 h. Moreover, we found a similar expression kinetic and trafficking in human monocyte derived MΦ. This places CD83 at a very early point of MΦ activation suggesting an important role in decisions regarding the subsequent cellular fate.

Identification of immunocell infiltrates and effective diagnostic biomarkers in laryngeal carcinoma.

Laryngeal cancer (LC) is a malignant tumor that occurs in the head and neck. Laryngeal cancer is one of the most common cancers of the neck and head, and its prognosis has always been poor. The incidence of LC increased gradually and showed an early rising trend. Laryngeal cancer is rarely studied in relation to immunity, Malignant tumors will change the state of the human body in various ways to adapt to their own survival and avoid the immune system. This study aims to explore the immune molecular mechanism of laryngeal cancer through bioinformatics analysis. The gene expression data was downloaded for 3 microarray datasets: GSE27020, GSE59102, and GSE51985. CIBERSORT algorithm was performed to evaluate immune cell infiltration in tissues between LC and healthy control (HC). Differentially expressed genes (DEGs) were screened. Functional correlation of DEGs were analyzed by Gene Ontology, Gene Set Enrichment Analysis and Kyoto encyclopedia of genes and genomes. Candidate biomarkers were identified by cytoHubba of Cytoscape. Spearman correlations between the above biomarkers and infiltrating immune cells were explored using R software analysis. The immune cell types of LC and HC were significantly different. Twenty-one DEGs were obtained by cross-screening. The function of DEGs is closely related to the number of immune cells. Five central genes (TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4) were screened. The HUB gene was demonstrated to have the ability to diagnose LC and HC with good specificity and sensitivity. The correlation between immune cells and biomarkers showed that hub gene was positively correlated with macrophages and dendritic cells, and negatively correlated with CD4 + T cell. TNNT3, TNNI2, Desmin, matrix metallopeptidase 9 and cytotoxic T lymphocyte antigen 4 can be used as diagnostic biomarker for LC. Macrophages, dendritic cells and CD4 + T cell may participate in the occurrence and development of LC.

The ligation of CD4 molecules, expressed on monocytes by an anti-CD4 monoclonal antibody, inhibits T cell activation and monocyte mobility.

CD4, a leukocyte surface glycoprotein, is mainly expressed on CD4+ T cells, but is also expressed on monocytes. The difference in the expression level and structure of CD4 on T cells and monocytes predicts the different functions of this molecule in both cell types. Although the function of CD4 on T cells is well characterized, little is known about that expressed on primary monocytes. In this study, we investigated the immunoregulation function of CD4 on peripheral blood monocytes. Methods: CD4 molecule on monocyte was ligated by anti-CD4 monoclonal antibody (mAb), MT4/3. The effect of mAb MT4/3 on T cell proliferation, cytokine production, the expression of monocyte costimulatory molecules, monocyte migration, and macrophage differentiation were investigated. Moreover, the molecular weight of CD4 on peripheral blood monocyte was carried out by Western immunoblotting. We demonstrated that mAb MT4/3 inhibited anti-CD3 induced T cell proliferation, cytokine production, and the expression of monocyte costimulatory molecules. The ligation of only CD4 on monocytes was sufficient to inhibit T cell activation. Moreover, mAb MT4/3 could inhibit monocyte migration in a transwell migration assay, but not affect the differentiation of monocytes to macrophages. Using purified primary monocytes, the molecular weight of CD4 expressed on monocytes was identified as 55 kDa. The CD4 molecule expressed on monocytes might play an important role in the regulation of immune responses in both innate and adaptive immunity. Understanding the novel role of CD4 on monocytes in immunoregulation is valuable in the development of new therapeutic approaches.

Characterization of intrahepatic B cells in acute-on-chronic liver failure.

Acute on chronic liver failure (ACLF) is characterized by the immunologic dissonance during the prolonged pathogenic development. Both abnormal innate immune response and adaptive T-cell response have been reported in patients with ACLF; however, less is known regarding B cells in ACLF pathogenesis. Previous reports were only based on immunophenotyping of peripheral blood samples. Here, we aim to dissect liver-infiltrating B-cell subpopulation in ACLF. Paired liver perfusate and peripheral blood were freshly collected from healthy living donors and recipients during liver transplantation. Liver tissues were obtained from patients with ACLF, cirrhosis, and healthy controls. Flow cytometry was used to characterize the phenotypic and functional alterations in intrahepatic and circulating B-cell populations from ACLF, cirrhosis, and healthy controls. The expression of CD19+ and CD138+ on liver tissues was examined by immunohistochemistry staining. In this study, we first deciphered the intrahepatic B cells subsets of patients with ACLF. We found that the ACLF liver harbored reduced fraction of naïve B cells and elevated percentage of CD27+CD21- activated memory B cells (AM), CD27-CD21- atypical memory B cells (atMBC), CD27+IgD-IgM+(IgM+ memory B cells), and CD27+CD38++ plasma cells than cirrhosis and healthy controls. Moreover, these B subpopulations demonstrated enhanced activation and altered effector functions. Specifically, the ACLF liver was abundant in atMBC expressing higher CD11c and lower CD80 molecule, which was significantly correlated to alanine aminotransferase and aspartate aminotransferase. In addition, we found that intrahepatic CD27+CD38++plasma cells were preferentially accumulated in ACLF, which expressed more CD273 (PD-L2) and secreted higher granzyme B and IL-10. Finally, the enriched hepatic plasma B cells were in positive association with disease severity indices including alkaline phosphatase and gamma-glutamyl transferase. In this pilot study, we showed an intrahepatic B-cell landscape shaped by the ACLF liver environment, which was distinct from paired circulating B-cell subsets. The phenotypic and functional perturbation in atMBC and plasma cells highlighted the unique properties of infiltrating B cells during ACLF progression, thereby denoting the potential of B-cell intervention in ACLF therapy.

Alternative polyadenylation events in epithelial cells sense endometritis progression in dairy cows

Endometritis (inflammation of the endometrial lining) is one of the most devastating reproductive diseases in dairy cattle, resulting in substantial production loss and causing more than $650 million in lost revenue annually in the USA. We hypothesize that alternative polyadenylation (APA) sites serve as decisive sensors for endometrium health and disease in dairy cows. Endometrial cells collected from 18 cows with purulent vaginal discharge scored 0 to 2 were used for APA profiling with our whole transcriptome termini site sequencing (WTTS-seq) method. Overall, pathogens trigger hosts to use more differentially expressed APA (DE-APA), more intronic DE-APA, more DE-APA sites per gene and more DE-genes associated with inflammation. Host CD59 molecule (CD59), Fc fragment of IgG receptor IIa (FCGR2A), lymphocyte antigen 75 (LY75) and plasminogen (PLG) may serve as initial contacts or combats with pathogens on cell surface, followed by activation of nuclear receptor subfamily 1 group H member 4 (NR1H4) to regulate AXL receptor tyrosine kinase (AXL), FGR proto-oncogene, Src family tyrosine kinase (FGR), HCK proto-oncogene, Src family tyrosine kinase (HCK) and integrin subunit beta 2 (ITGB2) for anti-inflammation. This study is the first to show significance of cilium pathways in endometrium health and animal reproduction. MIR21 and MIR30A would be perfect antagonistic biomarkers for diagnosis of either inflammation or anti-inflammation. These novel findings will set precedent for future genomic studies to aid the dairy industry develop new strategies to reduce endometritis incidence and improve fertility.

Construction of Dual- and Enhanced Dual-Antibodies Targeting CD19 in the Treatment of B-ALL and Investigation of the Relevant Mechanisms

Objective: Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by the development of immature lymphocytes. Although many patients can achieve complete remission (CR) after chemotherapy, only minority can sustain long term remission without relapse. In the past decade, chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses for B cells malignancies, however, it still encounters with substantial challenges, such as expensive cost and treatment failure. T cell-redirecting bispecific antibodies is able to build the connection between endogenous T cells and tumor cells in order to activate T cells function to eliminate cancer cells without the need for ex vivo genetic alteration or manipulation of the T cells. It can provide an off-the-shelf immune-oncology therapy, unlike the long-term preparation before CAR-T infusion. Here, we developed the novel dual-specific antibody (DuAb) and enhanced DuAb (EDuAb) with different stimulation signals to activate T cells and explored their impact on the treatment of hematologic tumors by altering the function of T cells. Methods: The expression plasmids of dual-specific antibody targeting CD19 and CD3 (DuAb) and the enhanced DuAb (EDuAb) containing CD80 molecule were constructed by cloning heavy chain and light chain variable fragments from previously established anti-human CD19 (HI19a) and CD3 (HIT3a) monoclonal antibodies, respectively. Both DuAb and EDuAb were produced through a eukaryotic expression system. The activation of human primary T cells was evaluated after stimulated by DuAb and EDuAb in vitro, by detecting T cells proliferation, cytokine secretion, apoptosis and inhibitor marker expression. The antitumor efficacy of human primary T cells mediated by DuAb and EDuAb was evaluated through co-culture killing assay. B-ALL xenograft NSG mouse model was established to investigate the in vivo therapeutic effect. Results: EDuAb promoted the optimal expansion of primary human T cells with low expression of inhibitor marker in vitro than DuAb did (fold change of proliferation on day 9: 61.39 vs 19.23, p<0.0001; proportion of annexin V+: 11.83% vs 45.13%, p<0.0001, Tim3+: 20.08% vs 35.37%, p=0.007, PD-1+: 11.6% vs 34.3%, p=0.001, Lag3+: 29.12% vs 51.42%, p=0.006). In addition, BCL-XL was upregulated and IL2 secretion increased in EDuAb group. DuAb and EDuAb showed a similar capability in inducing healthy donor T cells to specifically eliminate B-ALL cell lines in vitro. At the concentration of 0.01nM, E/T ratio (2/1), the lysis of Nalm6 cells exceeded 96% after co-cultured for 48 hours. The similar ability was also observed in the patient-derived T cells. Both DuAb and EDuAb markedly suppressed tumor burden to baseline level and extended survival of B-ALL xenograft NSG mice. The median survival of vehicle, DuAb and EDuAb treatment group were 27, 38 and 45 days, respectively, which illustrated that EDuAb had a better therapeutic effect in vivo than that of DuAb (p=0.0066). The phenotype of T cells and cytokine release in peripheral blood of B-ALL xenograft NSG mouse model on day 24 were analyzed as well. The results showed that the proportion of CD8+ T cells and the cytokine levels, including IL2, IFN-γ and TNF-α, were higher in the EDuAb group than that of DuAb (CD8+: 0.746% vs 0.064%, p=0.0079; IL2: 1.862pg/ml vs 0.654pg/ml，p=0.0014; IFN-γ: 432.27pg/ml vs 1.48pg/ml, p= 0.047; TNF-α: 4.57pg/ml vs 2.08pg/ml, p=0.018). Both DuAb and EDuAb could significantly decrease the residual Nalm6 cells in PB of B-ALL xenograft NSG mice, and the residual tumor cells on day 24 were 1.61%, 0.45% and 0.29% in Vehicle, DuAb and EDuAb groups, respectively (Vehicle vs DuAb, p=0.0134; Vehicle vs EDuAb, p=0.0034; DuAb vs EDuAb, p=0.0169). Conclusion: Both DuAb and EDuAb showed great potential for clinical application as a novel treatment method for B-ALL. Compared with DuAb, there is a significant advantage for EDuAb to promote the proliferation and survival of T cells. In addition, EDuAb showed better promising effect to eliminate tumor cells and extend survival in vivo, which provides a new insight for constructing new multi-specific antibodies.