Immune-mediated Clearance Research Articles

716 Restoration of hair follicle inductive properties by depletion of senescent cells

Under physiological conditions, senescent cells (SCs) attract immune cells through secretion of senescence-associated secretory phenotype (SASP), which facilitates immune-mediated clearance of SCs. If SCs are not efficiently cleared, SASP accumulation leads to pathologic dysfunction of nearby non-senescent cells. Human dermal papilla (DP) cells lose their original inductive properties when expanded in in vitro culture, in which senescent cells are never cleared without specific treatment. We discovered that human DP culture accumulated SCs over passaging, which exhibited enlarged nucleocytoplasmic morphology correlating with β-galactosidase and p21 expression.

Meta analysis of bioactive compounds, miRNA, siRNA and cell death regulators as sensitizers to doxorubicin induced chemoresistance.

Cancer has presented to be the most challenging disease, contributing to one in six mortalities worldwide. The current treatment regimen involves multiple rounds of chemotherapy administration, alone or in combination. The treatment has adverse effects including cardiomyopathy, hepatotoxicity, and nephrotoxicity. In addition, the development of resistance to chemo has been attributed to cancer relapse and low patient overall survivability. Multiple drug resistance development may be through numerous factors such as up-regulation of drug transporters, drug inactivation, alteration of drug targets and drug degradation. Doxorubicin is a widely used first line chemotherapeutic drug for a myriad of cancers. It has multiple intracellular targets, DNA intercalation, adduct formation, topoisomerase inhibition, iron chelation, reactive oxygen species generation and promotes immune mediated clearance of the tumor. Agents that can sensitize the resistant cancer cells to the chemotherapeutic drug are currently the focus to improve the clinical efficiency of cancer therapy. This review summarizes the recent 10-year research on the use of natural phytochemicals, inhibitors of apoptosis and autophagy, miRNAs, siRNAs and nanoformulations being investigated for doxorubicin chemosensitization.

Abstract 1380: Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance

Abstract While immunotherapies have been revolutionary for the treatment of many cancers including melanoma, most patients do not respond, or develop resistance to these therapies. Several epigenetic modulators have been identified as potential targets for augmenting anti-tumor immunity, however the full breadth of their role is not well-understood. To identify modulators of anti-tumor immunity in melanoma, we performed a whole-genome CRISPR screen using the YUMMER-G model and identified members of the HUSH complex and its associated methyltransferase, Setdb1, as drop-out hits. To validate these findings, we used CRISPR-Cas9 to knockout Setdb1 individually and observed that Setdb1-/- tumors are cleared by their host mice in a CD8+ T-cell dependent manner. To determine the source of immunogenicity in Setdb1-/- cells, we performed RNA-sequencing, cytokine profiling, and evaluated antigen presentation by flow cytometry. These assays revealed increased MHC-I levels, elevated secretion of Ifn-, and induction of an interferon-stimulated gene (ISG) expression program, consistent with the observed immunogenicity. We further determined that treating Setdb1-/- cells with IFNAR-blocking antibodies restores MHC-I levels to wild-type levels, suggestive of a tumor-intrinsic interferon loop. We hypothesized that type-I interferon signaling is generated through sensing of expressed endogenous retroviral elements (ERVs), and consistently observe upregulation of ERVs in Setdb1-/- cells. Previous studies have described the potential for ERV-derived antigens to act as substrates for anti-tumor activity by CD8+ T cells. We identified T cells in the tumor microenvironment of Setdb1-/- tumors that bind tetramer carrying the P15E peptide derived from ERV envelope. Together, these findings suggest that tumors lacking expression of Setdb1-/- upregulate ERV transcripts, driving expression of type-I interferons which generate an inflamed tumor microenvironment and potentiate efficient tumor clearance of Setdb1-/- tumors. Overall, this study emphasizes a critical role for regulators of ERV expression and type-I interferon expression, and suggests their potential as therapeutic targets for augmenting anti-cancer immune responses. Citation Format: Meaghan K. McGeary, William Damsky, Drew Daniels, Goran Micevic, Eric Song, Hua Jane Lou, Clotilde Calderwood, Sateja Paradkar, Akiko Iwasaki, David Calderwood, Benjamin Turk, Marcus Bosenberg. Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance [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 1380.

Alpha-Synuclein Autoimmune Decline in Prodromal Multiple System Atrophy and Parkinson's Disease.

Multiple-system trophy (MSA) and Parkinson’s Disease (PD) are both progressive, neurodegenerative diseases characterized by neuropathological deposition of aggregated alpha-synuclein (αSyn). The causes behind this aggregation are still unknown. We have reported aberrancies in MSA and PD patients in naturally occurring autoantibodies (nAbs) against αSyn (anti-αSyn-nAbs), which are important partakers in anti-aggregatory processes, immune-mediated clearance, and anti-inflammatory functions. To elaborate further on the timeline of autoimmune aberrancies towards αSyn, we investigated here the Immunoglobulin (Ig) affinity profile and subclass composition (IgG-total, IgG1-4 and IgM) of anti-αSyn-nAbs in serum samples from prodromal (p) phases of MSA and PD. Using an electrochemiluminescence competition immunoassay, we confirmed that the repertoire of high-affinity anti-αSyn-nAbs is significantly reduced in pMSA and pPD. Further, we demonstrated that pPD had increased anti-αSyn IgG-total levels compared to pMSA and controls, concordant with increased anti-αSyn IgG1 levels in pPD. Anti-αSyn IgG2 and IgG4 levels were reduced in pMSA and pPD compared with controls, whereas anti-αSyn IgG3 levels were reduced in pMSA compared to pPD and controls. The results indicate that the impaired reactivity towards αSyn occurs prior to disease onset. The apparent lack of high-affinity anti-αSyn nAbs may result in reduced clearance of αSyn, leading to aggregation of the protein. Thus, this study provides novel insights into possible causes behind the pathogenesis in synucleinopathies such as MSA and PD.

TTI-621-03: A phase I/II study of TTI-621 in combination with doxorubicin in patients with unresectable or metastatic high-grade leiomyosarcoma (LMS).

TPS11593 Background: Doxorubicin is a standard of care agent for patients with advanced soft tissue sarcoma, with a response rate of around 15%, progression-free survival of 5-7 months and cumulative cardiac toxicity that limits its use. TTI-621 is a recombinant soluble fusion protein that combines the N-terminal portion of human SIRPα (the binding domain for CD47) with the Fc region of human IgG1, generating a decoy receptor for CD47 on the surface of tumor cells that both over-rides CD47-mediated inhibition of phagocytosis and provides a pro-phagocytic stimulation. Many solid tumors express high levels of CD47 which is associated with poor prognosis, thought to be the result of CD47-mediated inhibition of macrophage phagocytosis and escape of immune-mediated clearance. Interruption of the CD47-SIRPα signaling pathway using monoclonal antibodies to CD47 has shown anti-tumor activity in animal models and in some early clinical trials. The combination of doxorubicin with CD47-targeted antibodies results in enhanced anti-tumor activity and increased macrophage-mediated cell killing in animal models and macrophage-mediated phagocytosis of cancer cell lines in vitro, suggesting that combining TTI-621 with doxorubicin might be more effective than doxorubicin alone in tumor types that express CD47 and have high numbers of macrophages, such as LMS. Thus, a Phase 1/2 study was initiated to evaluate this combination in patients with advanced soft tissue sarcoma, including LMS. Methods: TTI-621-03 is a Phase 1/2, open-label study of TTI-621 in combination with doxorubicin in patients with anthracycline-naïve disease. The Phase 1 dose escalation evaluates doses of TTI-621 (0.2 to 2.0 mg/kg) in combination with doxorubicin at 75 mg/m2 in patients with high-grade soft tissue sarcomas. Expansion cohorts will evaluate TTI-621 (0.2 and 2.0 mg/kg) with doxorubicin in patients with LMS, with pathology confirmed at a central laboratory. The primary goals of this study are evaluation of safety of TTI-621 administered in combination with standard-of-care doxorubicin and to further evaluate clinical activity (ORR, PFS, OS), safety, PK and patient-reported quality of life in the LMS subpopulation. The dose escalation portion of the study has been completed without DLT. Enrollment to the expansion portion of the study is underway. Clinical trial information: NCT04996004.

Molecular dynamic simulations reveal anti-SARS-CoV-2 activity of mitocurcumin by potentially blocking innate immune evasion proteins NSP3 and NSP16.

The coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is affecting human life in an unprecedented manner and has become a global public health emergency. Identification of novel inhibitors of viral infection/replication is the utmost priority to curtail COVID-19 progression. A pre-requisite for such inhibitors is good bioavailability, non-toxicity and serum stability. Computational studies have shown that curcumin can be a candidate inhibitor of certain SARS-CoV-2 proteins; however, poor bio-availability of curcumin limits its possible therapeutic application. To circumvent this limitation, we have used mitocurcumin (MC), a triphenyl phosphonium conjugated curcumin derivative, to study the ability to inhibit SARS-CoV-2 infection using molecular docking and molecular dynamics (MD) simulation. MC is serum stable and several fold more potent as compared to curcumin. Molecular docking studies revealed that MC can bind at active site of SARS-CoV-2 ADP Ribose Phosphatase (NSP3) and SARS-CoV-2 methyltransferase (NSP10-NSP16 complex) with a high binding energy of − 10.3 kcal/mol and − 10.4 kcal/mol, respectively. MD simulation (100 ns) studies revealed that binding of MC to NSP3 and NSP16 resulted in a stable complex. MC interacted with critical residues of NSP3 macro-domain and NSP10-NSP16 complex and occupied their active sites. NSP3 is known to suppress host immune responses whereas NSP10-NSP16 complex is known to prevent immune recognition of viral mRNA. Our study suggests that MC can potentially inhibit the activity of NSP3 and NSP10-NSP16 complex, resulting in compromised viral immune evasion mechanism, and thereby accentuate the innate immune mediated clearance of viral load.Graphical abstract

The Investigation of the Effect of Antibody Recruiting Molecules on Various Antigenic Markers (Cancer, Bacteria, Viruses): A Literature Review

Introduction: Antibody recruiting molecules (ARMs) are small molecules with low molecular weight that guide endogenous antibodies towards both cancer and infectious cells, they facilitate the process of immune-mediated clearance. ARMs have two specific regions; a Target Binding Terminus interacts with disease biomarkers and the Antibody Binding Terminus, associated with endogenous antibodies. These modules are linked together by a tunable linker region bridging the endogenous antibody and the infected cell. ARMS can be used for a broad range of therapeutic applications, especially for its use against cancer, bacterial, and viral infections. ARMs serve a new potential treatment option over traditional therapies. Methods: To conduct our research, specific search terms were created, and relevant articles were screened on Covidence using an inclusion/exclusion criteria. The CASP and CRAAP checklist will be used for the quality assessment of the utilized sources. Results: ARMs treatment is a novel pathway which can treat a wide range of diseases from cancer, bacteria, to viruses. ARMs clearly represent promising alternatives in antitumor immunotherapy over traditional methods. Discussion: One hurdle of using ARMs is that its effect on individuals might differ based on antibody concentrations, their affinities, isotypes etc. Due to the non-specific nature of ARMs, there’s a selectivity issue regarding binding to specific biomarkers or antigens. The use of non-covalent ARMs to target the highly expressed receptors on the tumor can sometimes lead to endocytosis during the binding process before the recruitment of antibodies. This can be potentially solved by adding covalent linkages in the ARMs molecular construct. This paper analyzes the limitations of utilizing ARMs as an effective means for immunotherapy and proposes potential avenues of improvement for greater efficacy. Conclusion: This paper will potentially advance pharmaceutical and immunotherapeutic interventions available for numerous cancers and infectious diseases.

Understanding T cell responses to COVID-19 is essential for informing public health strategies.

Durable T cell responses to SARS-CoV-2 antigens after infection or vaccination improve immune-mediated viral clearance. To date, population-based surveys of COVID-19 adaptive immunity have focused on testing for IgG antibodies that bind spike protein and/or neutralize the virus. Deployment of existing methods for measuring T cell immunity could provide a more complete profile of immune status, informing public health policies and interventions.

Crosstalk Between Senescent Bone Cells and the Bone Tissue Microenvironment Influences Bone Fragility During Chronological Age and in Diabetes.

Bone is a complex organ serving roles in skeletal support and movement, and is a source of blood cells including adaptive and innate immune cells. Structural and functional integrity is maintained through a balance between bone synthesis and bone degradation, dependent in part on mechanical loading but also on signaling and influences of the tissue microenvironment. Bone structure and the extracellular bone milieu change with age, predisposing to osteoporosis and increased fracture risk, and this is exacerbated in patients with diabetes. Such changes can include loss of bone mineral density, deterioration in micro-architecture, as well as decreased bone flexibility, through alteration of proteinaceous bone support structures, and accumulation of senescent cells. Senescence is a state of proliferation arrest accompanied by marked morphological and metabolic changes. It is driven by cellular stress and serves an important acute tumor suppressive mechanism when followed by immune-mediated senescent cell clearance. However, aging and pathological conditions including diabetes are associated with accumulation of senescent cells that generate a pro-inflammatory and tissue-destructive secretome (the SASP). The SASP impinges on the tissue microenvironment with detrimental local and systemic consequences; senescent cells are thought to contribute to the multimorbidity associated with advanced chronological age. Here, we assess factors that promote bone fragility, in the context both of chronological aging and accelerated aging in progeroid syndromes and in diabetes, including senescence-dependent alterations in the bone tissue microenvironment, and glycation changes to the tissue microenvironment that stimulate RAGE signaling, a process that is accelerated in diabetic patients. Finally, we discuss therapeutic interventions targeting RAGE signaling and cell senescence that show promise in improving bone health in older people and those living with diabetes.

Human Immunodeficiency Virus-1 Latency Reversal via the Induction of Early Growth Response Protein 1 to Bypass Protein Kinase C Agonist-Associated Immune Activation.

Human Immunodeficiency Virus-1 (HIV) remains a global health challenge due to the latent HIV reservoirs in people living with HIV (PLWH). Dormant yet replication competent HIV harbored in the resting CD4+ T cells cannot be purged by antiretroviral therapy (ART) alone. One approach of HIV cure is the “Kick and Kill” strategy where latency reversal agents (LRAs) have been implemented to disrupt latent HIV, expecting to eradicate HIV reservoirs by viral cytopathic effect or immune-mediated clearance. Protein Kinase C agonists (PKCa), a family of LRAs, have demonstrated the ability to disrupt latent HIV to an extent. However, the toxicity of PKCa remains a concern in vivo. Early growth response protein 1 (EGR1) is a downstream target of PKCa during latency reversal. Here, we show that PKCa induces EGR1 which directly drives Tat-dependent HIV transcription. Resveratrol, a natural phytoalexin found in grapes and various plants, induces Egr1 expression and disrupts latent HIV in several HIV latency models in vitro and in CD4+ T cells isolated from ART-suppressed PLWH ex vivo. In the primary CD4+ T cells, resveratrol does not induce immune activation at the dosage that it reverses latency, indicating that targeting EGR1 may be able to reverse latency and bypass PKCa-induced immune activation.

Experimental Polymorphism Survey in Intergenic Regions of the icaADBCR Locus in Staphylococcus aureus Isolates from Periprosthetic Joint Infections.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics. The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of polymorphisms in bacterial genomes may help to establish associations between sequence variants and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI.

Antigen Capture and Immune Modulation by Bacterial Outer Membrane Vesicles as In Situ Vaccine for Cancer Immunotherapy Post-Photothermal Therapy.

Tumor antigens released from tumor cells after local photothermal therapy (PTT) can activate the tumor-specific immune responses, which are critical for eliminating the residual lesions and distant metastases. However, the limited recognition efficiency of released tumor antigens by the immune system and the immunosuppressive microenvironment lead to ineffective antitumor immunity. Here, an in situ multifunctional vaccine based on bacterial outer membrane vesicles (OMVs, 1-MT@OMV-Mal) is developed by surface conjunction of maleimide groups (Mal) and interior loading with inhibitor of indoleamine 2, 3-dioxygenase (IDO), 1-methyl-tryptophan (1-MT). 1-MT@OMV-Mal can bind to the released tumor antigens after PTT, and be efficiently recognized and taken up by dendritic cells. Furthermore, in situ injection of 1-MT@OMV-Mal simultaneously overcomes the immune inhibition of IDO on tumor-infiltrating effector T cells, leading to remarkable inhibition on both primary and distant tumors. Together, a promising in situ vaccine based on OMVs to facilitate immune-mediated tumor clearance after PTT through orchestrating antigen capture and immune modulation is presented.

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice.

Acute otitis media (AOM) is commonly caused by bacterial pathobionts of the nasopharynx that ascend the Eustachian tube to cause disease in the middle ears. To model and study the various complexities of AOM, common human otopathogens are injected directly into the middle ear bullae of rodents or are delivered with viral co-infections which contribute to the access to the middle ears in complex and partially understood ways. Here, we present the novel observation that Bordetella bronchiseptica, a well-characterized respiratory commensal/pathogen of mice, also efficiently ascends their Eustachian tubes to colonize their middle ears, providing a flexible mouse model to study naturally occurring AOM. Mice lacking T and/or B cells failed to resolve infections, highlighting the cooperative role of both in clearing middle ear infection. Adoptively transferred antibodies provided complete protection to the lungs but only partially protected the middle ears, highlighting the differences between respiratory and otoimmunology. We present this as a novel experimental system that can capitalize on the strengths of the mouse model to dissect the molecular mechanisms involved in the generation and function of immunity within the middle ear.

The Mycobacterium tuberculosis PE_PGRS Protein Family Acts as an Immunological Decoy to Subvert Host Immune Response.

Mycobacterium tuberculosis (M.tb) is a successful pathogen that can reside within the alveolar macrophages of the host and can survive in a latent stage. The pathogen has evolved and developed multiple strategies to resist the host immune responses. M.tb escapes from host macrophage through evasion or subversion of immune effector functions. M.tb genome codes for PE/PPE/PE_PGRS proteins, which are intrinsically disordered, redundant and antigenic in nature. These proteins perform multiple functions that intensify the virulence competence of M.tb majorly by modulating immune responses, thereby affecting immune mediated clearance of the pathogen. The highly repetitive, redundant and antigenic nature of PE/PPE/PE_PGRS proteins provide a critical edge over other M.tb proteins in terms of imparting a higher level of virulence and also as a decoy molecule that masks the effect of effector molecules, thereby modulating immuno-surveillance. An understanding of how these proteins subvert the host immunological machinery may add to the current knowledge about M.tb virulence and pathogenesis. This can help in redirecting our strategies for tackling M.tb infections.

Fighting HIV-1 Persistence: At the Crossroads of "Shoc-K and B-Lock".

Despite the success of highly active antiretroviral therapy (HAART), integrated HIV-1 proviral DNA cannot be eradicated from an infected individual. HAART is not able to eliminate latently infected cells that remain invisible to the immune system. Viral sanctuaries in specific tissues and immune-privileged sites may cause residual viral replication that contributes to HIV-1 persistence. The “Shock or Kick, and Kill” approach uses latency reversing agents (LRAs) in the presence of HAART, followed by cell-killing due to viral cytopathic effects and immune-mediated clearance. Different LRAs may be required for the in vivo reactivation of HIV-1 in different CD4+ T cell reservoirs, leading to the activation of cellular transcription factors acting on the integrated proviral HIV-1 LTR. An important requirement for LRA drugs is the reactivation of viral transcription and replication without causing a generalized immune activation. Toll-like receptors, RIG-I like receptors, and STING agonists have emerged recently as a new class of LRAs that augment selective apoptosis in reactivated T lymphocytes. The challenge is to extend in vitro observations to HIV-1 positive patients. Further studies are also needed to overcome the mechanisms that protect latently infected cells from reactivation and/or elimination by the immune system. The Block and Lock alternative strategy aims at using latency promoting/inducing agents (LPAs/LIAs) to block the ability of latent proviruses to reactivate transcription in order to achieve a long term lock down of potential residual virus replication. The Shock and Kill and the Block and Lock approaches may not be only alternative to each other, but, if combined together (one after the other), or given all at once [namely “Shoc-K(kill) and B(block)-Lock”], they may represent a better approach to a functional cure.

Suppression of Extracellular Vesicle CD47 Induces Systemic Anti-DLBCL Immunity

Purpose:Tumor cells evade the immune surveillance by up-regulating surface expression of CD47, which interacts with SIRPa on macrophages to elicit the immune checkpoint response. Anti-CD47 antibody (IBI188) has shown promise in treating tumors, including Diffuse large B cell lymphoma (DLBCL). The objective response rate among DLBCL was 73% (ClinicalTrials.gov number, NCT02953509.). The basis of differential therapeutic success between patients remains unknown. Extracellular vesicles (EVs) carry bioactive molecules that influence the immune system. CD47 has be found on surface of EVs. The purpose of this study is to explore whether EV CD47 contributes to immunosuppression and is associated with anti-CD47 response in DLBCL.Methods: 1.Kaplan-Meier curves and Cox regression models were used to analyze PFS and OS. The area under the ROC curve (AUC) was used to assess the predicted validity of the National Comprehensive Cancer Network-International Prognostic Index (NCCN-IPI) model and the NCCN-IPI+ CD47 model.2.Immunofluorescence experiment was used to detect the infiltration of M1 and M2 subtype macrophages in DLBCL tumor tissue. M1 and M2 macrophages were generated from peripheral blood mononuclear cells obtained from healthy donors. Immunoelectron microscopy, flow cytometry and western blot were performed to detect CD47 on the surface of the EVs derived from DLBCL cells. The activity of macrophage phagocytosis of DLBCL cells was detected by confocal-based phagocytosis assay and flow cytometry-based phagocytosis assay.3.In DLBCL xenografts, the IBI188 antibody was a macrophage immune checkpoint inhibitor blocking CD47 that induces tumor-cell phagocytosis.4.Peripheral blood mononuclear cells from DLBCL patients and healthy donors was collected for EVs purification and subsequent detection of human CD47 proteins by ELISA.Results 1.The elevated expression of CD47 in DLBCL is significantly correlated with poor PFS and OS in a univariate analysis, and is statistically significant after adjusting for the NCCN-IPI in the univariate and multivariate analysis (Figure 1A, B). AUC analysis with cross-validation showed that the combination of the CD47 signature and NCCN-IPI had a better prediction of PFS and OS than without the CD47 signature (Figure 1C). Thus, CD47 in DLBCL can be used as a biomarker of prognosis and developed the prognostic stratification of DLBCL patients.2.EV CD47 derived from DLBCL cells has the same membrane topology as the cell surface CD47, with its extracellular domain exposed on the surface of the EVs (Figure 1D-G). Moreover, EV CD47 could function similarly to cell-surface CD47 in the suppression of macrophage phagocytosis of DLBCL cells (Figure 1H, I). DLBCL cells that had higher levels of cellular CD47 protein packaged greater amounts of CD47 into EVs, and these EVs displayed an increased binding to SIRPa of macrophages, thus inhibiting macrophage-mediated phagocytosis of DLBCL cells (Figure 1J-O).3.Antibodies against human CD47 specifically identified human CD47 on the circulating exosomes from mice bearing DLBCL xenografts but not the control mice. The level of circulating EV CD47 positively correlated with tumor size. Injection of EVs derived from DLBCL cells promoted the growth of tumors, whereas pre-treatment of the EVs with anti-CD47 antibodies (IBI188), but not IgG isotype or CD63-blocking antibodies, inhibited the effect (Figure 1P, Q).4.The presence of CD47 in the EVs isolated from the plasma of DLBCL patients, and the level of CD47 on the circulating EVs was significantly higher in DLBCL patients than that in healthy donors (Figure 1R, S). The receiver ROC curve shows that the level of circulating EV CD47 could distinguish DLBCL patients from healthy donors.ConclusionOur studies suggest that the EV CD47-SIRPa interaction may represent a critical mechanism by which DLBCL cells escape immune-mediated clearance. Our study also raises the possibility that disrupting the interaction between the EV CD47 and macrophage SIRPa is a mechanism in the CD47-SIRPa blockade-based therapies. Moreover, high levels of circulating EV CD47 would follow and correlate positively with the phagocytic activity of macrophages, and reflect the presence of a successful anti-tumor immunity elicited by the anti-CD47 therapy. Together, these findings show that EV CD47 represents an unexplored therapeutic target, which could overcome resistance to current CD47 antibody approaches. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Triple-negative breast cancer cells rely on kinase-independent functions of CDK8 to evade NK-cell-mediated tumor surveillance

Triple-negative breast cancer (TNBC) is an aggressive malignant disease that is responsible for approximately 15% of breast cancers. The standard of care relies on surgery and chemotherapy but the prognosis is poor and there is an urgent need for new therapeutic strategies. Recent in silico studies have revealed an inverse correlation between recurrence-free survival and the level of cyclin-dependent kinase 8 (CDK8) in breast cancer patients. CDK8 is known to have a role in natural killer (NK) cell cytotoxicity, but its function in TNBC progression and immune cell recognition or escape has not been investigated. We have used a murine model of orthotopic breast cancer to study the tumor-intrinsic role of CDK8 in TNBC. Knockdown of CDK8 in TNBC cells impairs tumor regrowth upon surgical removal and prevents metastasis. In the absence of CDK8, the epithelial-to-mesenchymal transition (EMT) is impaired and immune-mediated tumor-cell clearance is facilitated. CDK8 drives EMT in TNBC cells in a kinase-independent manner. In vivo experiments have confirmed that CDK8 is a crucial regulator of NK-cell-mediated immune evasion in TNBC. The studies also show that CDK8 is involved in regulating the checkpoint inhibitor programmed death-ligand 1 (PD-L1). The CDK8–PD-L1 axis is found in mouse and human TNBC cells, underlining the importance of CDK8-driven immune cell evasion in these highly aggressive breast cancer cells. Our data link CDK8 to PD-L1 expression and provide a rationale for investigating the possibility of CDK8-directed therapy for TNBC.

Construction of β2m knockout mice

The β2m (Beta-2-microglobin) gene encodes a non-glycosylated protein that functions as an important component of major histocompatibility complexⅠ(MHCⅠ) for antigen presentation. To evade immune mediated clearance, human tumors and pathogens have adopted different strategies, including loss of MHCⅠexpression. Appropriate animal models are essential for understanding the mechanisms underpinning the clinical treatment of tumor and other human diseases. We constructed β2m knockout mice using CRISPR/Cas9 gene editing tool through embryo microinjection. Subsequently, genotyping and phenotyping of knockout mice were performed by PCR, qPCR, and flow cytometry. Mice genotyping showed that the coding region of the target gene was absent in the knockout mice. Real time PCR showed that mRNA level of β2m was significantly downregulated. Flow cytometry showed that the proportions of CD8+ killer T cells was significantly reduced in a variety of tissues and organs of the immune system. Taken together, we have successfully constructed a strain of β2m knockout mice, which will facilitate subsequent in vivo study on the function and mechanism of the β2m gene.

Uncovering immune and senescent cell interactions in disease

In this backstory, Anil Bhushan explains how recent work from his group investigating the immune-mediated clearance of senescent cells may offer a unique route to the development of senolytic therapies. These findings appeared in the May 2021 issue of Med (https://www.cell.com/med/fulltext/S2666-6340(21)00163-X).

Dynamically linking influenza virus infection kinetics, lung injury, inflammation, and disease severity.

Influenza viruses cause a significant amount of morbidity and mortality. Understanding host immune control efficacy and how different factors influence lung injury and disease severity are critical. We established and validated dynamical connections between viral loads, infected cells, CD8+ T cells, lung injury, inflammation, and disease severity using an integrative mathematical model-experiment exchange. Our results showed that the dynamics of inflammation and virus-inflicted lung injury are distinct and nonlinearly related to disease severity, and that these two pathologic measurements can be independently predicted using the model-derived infected cell dynamics. Our findings further indicated that the relative CD8+ T cell dynamics paralleled the percent of the lung that had resolved with the rate of CD8+ T cell-mediated clearance rapidly accelerating by over 48,000 times in 2 days. This complimented our analyses showing a negative correlation between the efficacy of innate and adaptive immune-mediated infected cell clearance, and that infection duration was driven by CD8+ T cell magnitude rather than efficacy and could be significantly prolonged if the ratio of CD8+ T cells to infected cells was sufficiently low. These links between important pathogen kinetics and host pathology enhance our ability to forecast disease progression, potential complications, and therapeutic efficacy.