CD4 Molecules Research Articles

Binding Domain on Cd22 Molecules Contributing to the Biological Activity of T Cell-Engaging Bispecific Antibodies

Binding Domain on Cd22 Molecules Contributing to the Biological Activity of T Cell-Engaging Bispecific Antibodies

Novel Bent Conformation of CD4 Induced by HIV-1 Inhibitor Indirectly Prevents Productive Viral Attachment

GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen–deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4′s flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.

Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis.

BackgroundDrug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin’s lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression.MethodsImmunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH.ResultsWe demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues.ConclusionsOur findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.

Glucocorticoid-induced Changes in the Transcriptional Activity of Genes of the Innate and Adaptive Immune System in the Blood of Patients with Acute Urticaria

Background: A number of the main effects of glucocorticoids (GCs) are their direct action on T cells, mainly through the transcriptional regulation: elevated expression of immune-regulatory proteins, inhibitory receptors, and reduced expression of pro-inflammatory cytokines, co-stimulatory molecules, and cell cycle mediators. But controversies arise due to the clinical effectiveness of GCs in the treatment of acute urticaria. Methods: In our research, we applied a pathway-specific PCR array (Human Innate & Adaptive Immune Responses RT2 Profiler PCR Array, QIAGEN, Germany) to detect and verify innate & adaptive immune responses pathway-focused genes expression in the blood of patients with acute urticaria who received treatment with glucocorticoids in addition to standard therapy. Results: Adding glucocorticoids to standard therapy did not notably affect the nature of the clinical presentation of acute urticaria, which was assessed according to the UAS scale (urticaria activity score). Analysis of the transcriptional profile of peripheral blood mononuclear cells in patients with acute urticaria against the background of glucocorticoid therapy showed the induction expression of the FOXP3 and IL10 genes against the background of repression of the transcriptional activity of the genes for chemokines and cytokines CCL5, CXCL8, IFNG, IL2, IL5, IL17A, IL1B, and TNF. Glucocorticoid-induced changes in the transcriptome also manifested by pronounced repression in genes of CD40 and CD80 (B7-1) co-stimulatory molecules, transcriptional regulators of Th1-cells differentiation - TBX21 and STAT1, Th17 cells - RORC, NLRP3-inflammasome genes, and the transcription factor NFKB1 compared with the control group. Conclusions: Adding glucocorticoids to the standard therapy of acute urticaria has a pronounced immunosuppressive potential at the transcriptome level of immune response genes in the blood; however, it does not have any noticeable clinical effect.

A High Sensitivity aCD22 CAR Combined with aCD19 CAR to Generate Dual Targeting CAR T Cells for the Treatment of r/r B-ALL

Background:Anti-CD19 CAR treatment has shown a remarkable response in r/r B-ALL patients, however, a major cause for relapse following CAR administration is the loss of the CD19 antigen. CD22 is a promising alternative target for B-ALL malignancies, although generating an effective CAR to CD22 is challenging due to the size, density and rigidity of this ligand. Furthermore, in the B-ALL setting the CD22 expression levels are known to down regulate in response to selective CAR pressure. This resulted in patients relapsing with lower CD22 density post-treatment (2,839 epitopes/cell) presumably due to the target density falling below the sensitivity threshold for the aCD22 CAR 1. In this study we aim to improve CAR efficacy for the treatment of r/r B-ALL by firstly developing a highly sensitive aCD22 CAR capable of targeting cells that express less than 1000 CD22 molecules per cell. Secondly, we combined this new aCD22 CAR with an aCD19 CAR to generate a dual targeting CD19 and CD22 product via co-transduction for the treatment of paediatric r/r B-ALL as part of an extension cohort of the CARPALL clinical trial (NCT02443831).Results:We screened 18 anti-CD22 binders with a range of affinities (1-36.5nM) and binding epitopes in CAR format for high functional sensitivity to low density targets. We filtered binders for their ability to maintain cytotoxicity, proliferation and cytokine release with low expressing CD22 targets. We identified the optimal aCD22 binder (9A8) which when combined in a CAR structure containing a 41BBz endodomain was capable of lysing target cells transduced to express less than 255 CD22 mols/cell while not affecting CD22 negative targets. Similar to the previously tested aCD22 binder M971, our new 9A8 binder also targets the membrane proximal Ig domain of the CD22 molecule. However unlike M971, 9A8 utilises a conventional 16 amino acid linker between the heavy and light chain to form the scFv and has a lower propensity to tonically signal compared to the M971 CAR. To generate a dual CD19 and CD22 targeting product, the 9A8 aCD22 CAR was co-transduced with the aCD19 CAT19 CAR vector (AUTO1) utilised in the CARPALL clinical trial for the treatment of paediatric r/r B-ALL. In a NSG animal model engrafted with WT CD19+/CD22+ Nalm-6 cells, the dual targeting co-transduced T cells (AUTO1/22) were significantly better at supressing tumour growth compared to single targeting AUTO1 CAR T cells (Figure 1). Furthermore, CAR T cells recovered from these animals showed an equal persistence of both aCD19 CAR and aCD22 CAR. In an effort to recapitulate the loss of CD19 target observed in aCD19 CAR relapsed patients, we challenged our transduced T cells in a CD19-knockout Nalm-6 NSG model (Figure 1). In this setting, single transduced AUTO1 CAR T cells were unable to control tumour growth above the non-transduced T cell controls. In contrast, the dual targeting co-transduced AUTO1/22 CAR T cells significantly supressed tumour growth and showed enrichment of CAR T cells expressing the aCD22 9A8 CAR in the bone marrow.The dual targeting co-transduced AUTO1/22 is currently being evaluated in an extension cohort of the CARPALL clinical trial (NCT02443831) for the treatment of paediatric r/r B-ALL.Conclusion:We engineered a highly sensitive aCD22 9A8 CAR which when combined with the aCD19 CAT19 CAR allows for improved in vivo function and the targeting of CD19 negative, CD22 positive cells. The use of these dual targeting T cells may reduce the incidence of CD19 negative relapses in the r/r B-ALL setting.1. Fry TJ, Shah NN, Orentas RJ, et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat. Med. 2018;24(1):20-28. [Display omitted] DisclosuresKokalaki: Autolus Ltd: Current Employment. Grothier: Autolus Ltd: Ended employment in the past 24 months. Manzoor: Autolus Ltd: Ended employment in the past 24 months. Costu: Autolus Ltd: Ended employment in the past 24 months. Srivastava: Autolus Ltd: Current Employment. Jha: Autolus Ltd: Current Employment. Gealy: Autolus Ltd: Current Employment. Stanczuk: Autolus Ltd: Ended employment in the past 24 months. Robson: Autolus Ltd: Current Employment. Taylor: Autolus Ltd: Current Employment. El-Kholy: Autolus Ltd: Current Employment. Baldan: Autolus Ltd: Current Employment. Righi: Autolus Ltd: Current Employment. Sillibourne: Autolus Ltd: Current Employment. Onuoha: Autolus: Ended employment in the past 24 months. Ghorashian: UCLB: Patents & Royalties; Novartis: Honoraria. Cordoba: Autolus Ltd: Current Employment. Amrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Pule: Autolus Ltd: Current Employment.

Anti-FLT3 CAR T Cells in Acute Myeloid Leukemia

INTRODUCTIONIn patients with AML who are eligible for intensive therapy, the goal of treatment is the achievement of complete response followed by consolidation chemotherapy (in favorable risk disease) or hematopoietic stem cell transplantation (in intermediate or adverse risk disease). Patients who do not attain this initial goal lack effective therapeutic options. Extensive experience with chimeric antigen receptor (CAR) T cells in B-ALL has shown that CART cells can deliver potent and durable antigen-specific leukemia control, and that targeting a single antigen (CD19 for B-ALL) is associated with antigen-negative relapse. In this context, we sought to expand the existing preclinical CART armamentarium in AML by developing FLT3-specific CART cells and comparing them to our existing gold standard CD123-specific CART cells. Since activating mutations in FLT3 occur commonly in AML, we reasoned that this molecule would serve as an “Achilles heel” in AML immunotherapy.METHODSNovel fully humanized anti-human FLT3 receptor single chain variable fragments (scFV) were fused to CD28 and CD137 (41BB) costimulatory molecules and the CD3zeta signaling domain and cloned into a lentiviral expression vector. Based on recently published data, we tested linker lengths ranging from 5 to 20 amino acids between the light and heavy chains of the CAR. We used a FLT3-ITD mutated AML cell line (MOLM14) expressing luciferase for in vitro function studies including an exhaustion assay. For in vivo function studies, we engrafted MOLM14 expressing luciferase into NSG mice and treated with CART-FLT3 or untransduced T cells (negative control).RESULTSAll FLT3 and CD123-specific CART cells degranulated and produced the effector cytokines IL-2, INFg, TNF and GM-CSF in an antigen-specific manner, with some variability between the different linker lengths and with some superiority of the CAR123 likely resulting from the higher expression of CD123 compared with FLT3 in this model (p < 0.0001, one way ANOVA) (Figure 1). Short-term killing assays (24 hours) revealed that all CART cells killed MOLM14 with equivalent efficiency at low effector:target ratios (Figure 2A). Since short-term killing assays likely do not replicate the physiological situation in vivo wherein CART cells encounter cancer cells repeatedly over many days, we next developed an in vitro exhaustion assay. We incubated MOLM14 cells with CAR T cells at 1:10 E:T ratio and added MOLM 14 tumor cells along with fresh media every other day. Killing was quantified every 48 hours. Interestingly, all CAR constructs showed equivalently efficient cytotoxicity from days 5-15. However, after day 15 there was progressive dysfunction and loss of cytotoxic activity. This exhaustion “stress test” revealed some superiority of the FLT3 CAR 10AA construct (p = 0.042 on day 17, two way ANOVA) (Figure 2B).NOD/SCID gamma chain KO (NSG) mice were then engrafted with 1x10 6 luciferized MOLM14 cells and treated with 0.5x10 6 CAR T cells 7 days later, randomized to treatment groups based on tumor burden. CAR T cells expansion was monitored in peripheral blood by flow cytometry. (Fig 3A). Serial BLI revealed prompt and durable leukemia remissions and survival (Figure 3B,C).CONCLUSIONSWe have developed CART-FLT3 for AML using novel human anti-FLT3 targeting domains and demonstrated preclinical efficacy similar to that of CART-123 in an AML model with substantially lower expression of FLT3 compared to CD123 (data not shown). Since inhibition of FLT3 leads to upregulation of surface FLT3 expression, future experiments will explore combinatorial FLT3 inhibition with CART-FLT3. If successful, these experiments will provide a strong rationale for a combination clinical trial in AML where leukemia control by small molecules is followed by a coup-de-grace delivered by CART cells. [Display omitted] DisclosuresSleiman: Hemogenyx Pharmaceuticals LLC: Research Funding. Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Santiago: Hemogenyx Pharmaceuticals LLC: Research Funding. Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang: Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda: Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler: Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Enhancement of the Local CD8+ T-Cellular Immune Response to Mycobacterium tuberculosis in BCG-Primed Mice after Intranasal Administration of Influenza Vector Vaccine Carrying TB10.4 and HspX Antigens.

BCG is the only licensed vaccine against Mycobacterium tuberculosis (M.tb) infection. Due to its intramuscular administration route, BCG is unable to induce a local protective immune response in the respiratory system. Moreover, BCG has a diminished ability to induce long-lived memory T-cells which are indispensable for antituberculosis protection. Recently we described the protective efficacy of new mucosal TB vaccine candidate based on recombinant attenuated influenza vector (Flu/THSP) co-expressing TB10.4 and HspX proteins of M.tb within an NS1 influenza protein open reading frame. In the present work, the innate and adaptive immune response to immunization with the Flu/THSP and the immunological properties of vaccine candidate in the BCG-prime → Flu/THSP vector boost vaccination scheme are studied in mice. It was shown that the mucosal administration of Flu/THSP induces the incoming of interstitial macrophages in the lung tissue and stimulates the expression of co-stimulatory CD86 and CD83 molecules on antigen-presenting cells. The T-cellular immune response to Flu/THSP vector was mediated predominantly by the IFNγ-producing CD8+ lymphocytes. BCG-prime → Flu/THSP vector boost immunization scheme was shown to protect mice from severe lung injury caused by M.tb infection due to the enhanced T-cellular immune response, mediated by antigen-specific effector and central memory CD4+ and CD8+ T-lymphocytes.

The Outcome of COVID-19 in Patients with a History of Taking Rituximab: A Narrative Review.

Coronavirus disease 2019 (COVID-19) is a recently emerging disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Notably, the safety of immunosuppressive medications is a major concern during an infectious disease pandemic. Rituximab (RTX), as a monoclonal antibody against CD20 molecule, is widely used for the treatment of various diseases, mostly autoimmune diseases and some malignancies. Previous studies indicated that RTX, as an immunosuppressive medication, may be associated with the increased risk of infections. Moreover, given the wide use of RTX, a necessity of determining the different aspects of RTX use in the COVID-19 era is strongly felt. We reviewed current studies on the clinical courses of patients with SARS-CoV-2 infection. It appears that the use of RTX does not increase morbidity and mortality in most patients. However, underlying diseases and other concomitant medications may play a role in the disease course, while the concerns of vaccine efficacy in patients receiving RTX still need to be addressed. Therefore, more controlled studies are needed for a better conclusion.

Orientation Dynamics of the Methyl Radical and Matrix Molecules in Solid Methane, CH4, and CD4 at Cryogenic Temperatures

Orientation Dynamics of the Methyl Radical and Matrix Molecules in Solid Methane, CH4, and CD4 at Cryogenic Temperatures

Anti‐B cell therapies in multiple sclerosis

AbstractMultiple sclerosis (MS) has long been considered a T‐cell‐mediated autoimmune disease of the central nervous system. However, newer evidence shows that B cells play a crucial role in the pathogenesis of MS via antibody production, antigen presentation, T cell stimulation and activation, and production of pro‐inflammatory cytokines. Developing B cells express the CD20 molecule from the pre‐B cell to the plasmablast stage, and the anti‐CD20 monoclonal antibodies rituximab, ocrelizumab, ofatumumab and ublituximab are either in use or are being developed for treatment of MS as B cell‐depleting therapies. They have shown profound efficacy in strongly suppressing relapse and radiological disease activity. Another promising approach is the inhibition of Bruton’s tyrosine kinase (BTK), a key enzyme that is intricately involved in signaling from the B cell antigen receptor and the Fc receptors in B cells and myeloid cells. BTK is considered to play an essential role for differentiation and survival of B‐lineage cells, and BTK inhibition may dampen not only B cell activation but also innate immune activation in MS. Trials of BTK inhibitors are ongoing to establish their efficacy and safety in relapsing and progressive MS. The main safety issue with persistent B cell depletion is an increased risk of infection, possibly including an increased risk of severe COVID‐19; unfortunately, vaccine responses against COVID‐19 may also be blunted by anti‐CD20 monoclonal antibodies.

Diminished Peripheral CD29hi Cytotoxic CD4+ T Cells Are Associated With Deleterious Effects During SIV Infection.

Cytotoxic CD4+ T cells (CD4+ CTLs) limit HIV pathogenesis, as evidenced in elite controllers (a subset of individuals who suppress the virus without the need for therapy). CD4+ CTLs have also been shown to kill HIV-infected macrophages. However, little is known about their contribution towards HIV persistence, how they are affected following exposure to immune modulators like morphine, and what factors maintain their frequencies and function. Further, the lack of robust markers to identify CD4+ CTLs in various animal models limits understanding of their role in HIV pathogenesis. We utilized various PBMC samples obtained from SIV infected and cART treated rhesus macaques exposed to morphine or saline and subjected to flow cytometry evaluations. Thereafter, we compared and correlated the expression of CD4+ CTL-specific markers to viral load and viral reservoir estimations in total CD4+ T cells. We found that CD29 could be reliably used as a marker to identify CD4+ CTLs in rhesus macaques since CD29hi CD4+ T cells secrete higher cytotoxic and proinflammatory cytokines following PMA/ionomycin or gag stimulation. In addition, this immune cell subset was depleted during untreated SIV infection. Strikingly, we also observed that early initiation of cART reconstitutes depleted CD29hi CD4+ T cells and restores their function. Furthermore, we noted that morphine exposure reduced the secretion of proinflammatory cytokines/cytotoxic molecules in CD29hi CD4+ T cells. Lastly, increased functionality of CD29hi CD4+ T cells as depicted by elevated levels of either IL-21 or granzyme B hi T Bet+ gag specific responses were linked to limiting the size of the replication-competent reservoir during cART treatment. Collectively, our data suggest that CD4+ CTLs are crucial in limiting SIV pathogenesis and persistence.

Activation of Toll-like receptor 2 enhances peripheral and tumor-infiltrating CD8+ T cell cytotoxicity in patients with gastric cancer

BackgroundToll-like receptors (TLRs) play central roles in the initiation of innate immune response, and also control adaptive immunity activation. Thus, the aim of the study was to investigate the regulation of TLR activation to CD8+ T cells has not been fully elucidated in gastric cancer (GC).Materials and methodsThirty-two GC patients and twenty-three healthy controls were enrolled. Expression profile of TLRs in peripheral and tumor-infiltrating CD8+ T cells was investigated. Purified CD8+ T cells were stimulated with Pam3Csk4, an agonist of TLR2, and cytotoxic and co-inhibitory molecules in CD8+ T cells was measured. Direct and indirect contact coculture system between CD8+ T cells and AGS cells was set up. Modulation of TLR2 activation to CD8+ T cells was assessed by measuring lactate dehydrogenase release and cytokine secretion.ResultsTLR2 mRNA and TLR2+ cell percentage was down-regulated in GC derived peripheral and tumor-infiltrating CD8+ T cells. CD8+ T cells from GC patients showed exhausted phenotype, which presented as decreased perforin/granzyme B, increased programmed death-1, and reduced cytotoxicity to AGS cells. TLR2 activation by Pam3Csk4 enhanced perforin and granzyme B expression in CD8+ T cells, however, did not affect either proinflammatory cytokine production or co-inhibitory molecules expression. Pam3Csk4 stimulation enhanced cytolytic activation of peripheral and tumor-infiltrating CD8+ T cells from GC, but not those from healthy individuals.ConclusionThe present data revealed an important immunomodulatory activity of TLR2 to CD8+ T cells in GC patients.

CD127+ CD94+ innate lymphoid cells expressing granulysin and perforin are expanded in patients with Crohn\u2019s disease

Phenotypic definition of helper ILC1 and NK cells is problematic due to overlapping markers. Recently we showed the identification of cytotoxic ILC3s characterized by expression of CD94. Here we analyse CD127+ ILCs and NK cells in intestinal lamina propria from healthy donors and Crohn’s disease patients and identify two populations of CD127+CD94+ ILCs, designated population A and B, that can be distinguished on the expression of CD117, CD18 and cytotoxic molecules. Population B expresses granulysin, a cytotoxic molecule linked to bacterial lysis and/or chemotaxis of monocytes. Granulysin protein is secreted by population B cells upon stimulation with IL-15. Activation of population B in the presence of TGF-β strongly reduces the expression of cytotoxic effector molecules of population B. Strikingly, samples from individuals that suffer from active Crohn’s disease display enhanced frequencies of granulysin-expressing effector CD127+CD94+ ILCs in comparison to controls. Thus this study identifies group 1 ILC populations which accumulate in inflamed intestinal tissue of Crohn’s disease patients and may play a role in the pathology of the disease.

Comparative microsomal proteomics of a model lung cancer cell line NCI-H23 reveals distinct differences between molecular profiles of 3D and 2D cultured cells.

Lung cancer is the leading cause of cancer-related deaths in the USA and worldwide. Yet, about 95% of new drug candidates validated in preclinical phase eventually fail in clinical trials. Such a high attrition rate is attributed mostly to the inability of conventional two-dimensionally (2D) cultured cancer cells to mimic native three-dimensional (3D) growth of malignant cells in human tumors.To ascertain phenotypical differences between these two distinct culture conditions, we carried out a comparative proteomic analysis of a membrane fraction obtained from 3D- and 2D-cultured NSCLC model cell line NCI-H23. This analysis revealed a map of 1,166 (24%) protein species regulated in culture dependent manner, including differential regulation of a subset of cell surface-based CD molecules. We confirmed exclusive expression of CD99, CD146 and CD239 in 3D culture. Furthermore, label-free quantitation, targeting KRas proteoform-specific peptides, revealed upregulation of both wild type and monoallelic KRas4BG12C mutant at the surface of 3D cultured cells.In order to reduce the high attrition rate of new drug candidates, the results of this study strongly suggests exploiting base-line molecular profiling of a large number of patient-derived NSCLC cell lines grown in 2D and 3D culture, prior to actual drug candidate testing.

Epicutaneous Administration of 17β-Estradiol Induces Langerhans Cells Depletion

ABSTRACT Background Langerhans cells (LC) number and function in mouse vaginal mucosa are affected by 17β-estradiol (E2) application; nonetheless, its effect on epidermal LC has not been studied. The purpose of this study was to evaluate the effect of topical administration of E2 on the number, phenotype, and migratory ability of LC in mouse skin. Methods Ears of adult CD1 male mice were topically treated once with several doses. Immunohistochemical staining for CD207 and TUNEL staining were performed. LC migration to lymph nodes and the effect on the expression of costimulatory molecules on cultured dendritic cells (DC) were also evaluated. Results E2 decreased the number of CD207+ LC in a dose-dependent manner. One hour after treatment, 1 and 10 µg/mL E2 significantly reduced the LC number by 21% and 26%, respectively, after two hours, the reduction was 23% and 41%, respectively. After 48 hours, LC recovered, and after 96 hours of treatment, the CD207+/MHCII+ DC numbers were increased in regional lymph nodes. However, CD86 and CD40 molecules were expressed at lower levels than in positive control. The TUNEL assay did not show apoptotic cells. Furthermore, in cultured DC, E2 promoted a decrease in CD40 and CD86 expression and an increase in CD273, CD274, MHCII, and CCR7. Conclusions The topical administration of E2 induced a transitory local diminution of LC population and a tolerogenic phenotype. This decrease in epidermal LC suggests that E2 may affect skin immune responses, inducing an inhibitory response, which should be considered when prescribing topical E2 medications.

Potential Molecular Mechanisms of Rare Anti-Tumor Immune Response by SARS-CoV-2 in Isolated Cases of Lymphomas.

Recently, two cases of complete remission of classical Hodgkin lymphoma (cHL) and follicular lymphoma (FL) after SARS-CoV-2 infection were reported. However, the precise molecular mechanism of this rare event is yet to be understood. Here, we hypothesize a potential anti-tumor immune response of SARS-CoV-2 and based on a computational approach show that: (i) SARS-CoV-2 Spike-RBD may bind to the extracellular domains of CD15, CD27, CD45, and CD152 receptors of cHL or FL and may directly inhibit cell proliferation. (ii) Alternately, upon internalization after binding to these CD molecules, the SARS-CoV-2 membrane (M) protein and ORF3a may bind to gamma-tubulin complex component 3 (GCP3) at its tubulin gamma-1 chain (TUBG1) binding site. (iii) The M protein may also interact with TUBG1, blocking its binding to GCP3. (iv) Both the M and ORF3a proteins may render the GCP2-GCP3 lateral binding where the M protein possibly interacts with GCP2 at its GCP3 binding site and the ORF3a protein to GCP3 at its GCP2 interacting residues. (v) Interactions of the M and ORF3a proteins with these gamma-tubulin ring complex components potentially block the initial process of microtubule nucleation, leading to cell-cycle arrest and apoptosis. (vi) The Spike-RBD may also interact with and block PD-1 signaling similar to pembrolizumab and nivolumab- like monoclonal antibodies and may induce B-cell apoptosis and remission. (vii) Finally, the TRADD interacting “PVQLSY” motif of Epstein-Barr virus LMP-1, that is responsible for NF-kB mediated oncogenesis, potentially interacts with SARS-CoV-2 Mpro, NSP7, NSP10, and spike (S) proteins, and may inhibit the LMP-1 mediated cell proliferation. Taken together, our results suggest a possible therapeutic potential of SARS-CoV-2 in lymphoproliferative disorders.

A siRNA-Assisted Assembly Strategy to Simultaneously Suppress "Self" and Upregulate "Eat-Me" Signals for Nanoenabled Chemo-Immunotherapy.

Effectively activating macrophages that can engulf cancer cells is a promising immunotherapeutic strategy but remains a major challenge due to the expression of "self" signals (e.g., CD47 molecules) by tumor cells to prevent phagocytosis. Herein, we explored a siRNA-assisted assembly strategy for the simultaneous delivery of siRNA and mitoxantrone hydrochloride (MTO·2HCl) via PLGA-based nanoparticles. The siRNA suppressed a "self" signal by silencing the CD47 gene, while the MTO induced surface exposure of calreticulin (CRT) to provide an "eat-me" signal. The siRNA-assisted assembly strategy synergistically increased the phagocytosis of tumor cells by macrophages, promoted effective antigen presentation, and initiated T cell-mediated immune responses in two aggressive tumor animal models of melanoma and colon cancer, eventually achieving significantly improved antitumor activity. This study provides a straightforward codelivery strategy to simultaneously suppress "self" and upregulate "eat-me" signals to potentiate macrophage-mediated immunotherapy.

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT

T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis, showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.

Sudden infant death syndrome: Melatonin, serotonin, and CD34 factor as possible diagnostic markers and prophylactic targets.

Sudden infant death syndrome (SIDS) is one of the primary causes of death of infants in the first year of life. According to the WHO’s data, the global infant mortality rate is 0.64–2 per 1,000 live-born children. Molecular and cellular aspects of SIDS development have not been identified so far. The purpose of this paper is to verify and analyze the expression of melatonin 1 and 2 receptors, serotonin (as a melatonin precursor), and CD34 molecules (as hematopoietic and endothelial markers of cardiovascular damage) in the medulla, heart, and aorta in infants who died from SIDS. An immunohistochemical method was used to investigate samples of medulla, heart, and aorta tissues of infants 3 to 9 months of age who died from SIDS. The control group included children who died from accidents. It has been shown that the expression of melatonin receptors as well as serotonin and CD34 angiogenesis markers in tissues of the medulla, heart, and aorta of infants who died from SIDS is statistically lower as compared with their expression in the same tissues in children who died from accidents. The obtained data help to clarify in detail the role of melatonin and such signaling molecules as serotonin and CD34 in SIDS pathogenesis, which can open new prospects for devising novel methods for predictive diagnosis of development and targeted prophylaxis of SIDS.