Generation and functional evaluation of novel monoclonal antibodies targeting glycosylated human stem cell factor.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.

Neurexophilin 4 is a prognostic biomarker correlated with immune infiltration in bladder cancer.

Recent studies have shown that NXPH family member 4 (NXPH4) plays an important role in the progression of cancer. However, the potential role of NXPH4 in bladder cancer (BCa) remains to be explored. The purpose of the present study was to identify whether NXPH4 could be used as a biomarker to predict the prognosis of BCa. We first examined the expression of NXPH4 in pan-cancer, and then focused on BCa. Univariate and multivariate Cox regression analysis were used to investigate whether NXPH4 could be used as an independent prognostic indicator. Gene set enrichment analysis (GSEA) was used for functional analysis of NXPH4-related genes. CIBERSORT algorithm was used to calculate immune cell infiltration levels with different NXPH4 expression. Finally, the expression of NXPH4 was validated in clinical tissue specimens and bladder cancer cell lines by immunohistochemistry and qRT-PCR. The tumor-promoting effects of NXPH4 were further investigated using counting kit-8 (CCK-8), colony formation, EdU assays, and tumor xenograft model. Our results showed that NXPH4 was highly expressed in BCa tissues. Patients in the high NXPH4 expression group had shorter overall survival (OS) and progression-free survival (PFS). We found that immune-related pathways were enriched in NXPH4-related genes. Immune cell infiltrations in BCa were also associated with different NXPH4 expression. NXPH4 was further found to be highly expressed in our validation specimens. The proliferative effect of NXPH4 was confirmed in BCa in vivo and in vitro. Overall, NXPH4 is a biomarker for predicting BCa prognosis and associated with immune infiltration.Abbreviations: NXPH4: Neurexophilin 4; BCa: Bladder cancer; TCGA-BLCA: The Cancer Genome Atlas Urothelial Bladder Carcinoma; shRNA: short hairpin RNA; NC: Negative control; OS: Overall survival; PFS: Progression-free survival; TME: Tumor microenvironment; IPS: immunophenoscore; ICIs: Immune checkpoint inhibitors; DEGs: Differential expression genes.

Immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccination in exposed and potentially exposed persons in the Democratic Republic of the Congo.

Despite more than 300,000 rVSVΔG-ZEBOV-glycoprotein (GP) vaccine doses having been administered during Ebola virus disease (EVD) outbreaks in the Democratic Republic of the Congo (DRC) between 2018 and 2020, seroepidemiologic studies of vaccinated Congolese populations are lacking. This study examines the antibody response at 21 d and 6 mo postvaccination after single-dose rVSVΔG-ZEBOV-GP vaccination among EVD-exposed and potentially exposed populations in the DRC. We conducted a longitudinal cohort study of 608 rVSVΔG-ZEBOV-GP-vaccinated individuals during an EVD outbreak in North Kivu Province, DRC. Participants provided questionnaires and blood samples at three study visits (day 0, visit 1; day 21, visit 2; and month 6, visit 3). Anti-GP immunoglobulin G (IgG) antibody titers were measured in serum by the Filovirus Animal Nonclinical Group anti-Ebola virus GP IgG enzyme-linked immunosorbent assay. Antibody response was defined as an antibody titer that had increased fourfold from visit 1 to visit 2 and was above four times the lower limit of quantification at visit 2; antibody persistence was defined as a similar increase from visit 1 to visit 3. We then examined demographics for associations with follow-up antibody titers using generalized linear mixed models. A majority of the sample, 87.2%, had an antibody response at visit 2, and 95.6% demonstrated antibody persistence at visit 3. Being female and of young age was predictive of a higher antibody titer postvaccination. Antibody response and persistence after Ebola vaccination was robust in this cohort, confirming findings from outside of the DRC.

Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.

C1q/tumour necrosis factor-related protein-9 aggravates lipopolysaccharide-induced inflammation via promoting NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a vital role in inflammation by increasing the maturation of interleukin-1ß (IL-1ß) and promoting pyroptosis. Given that C1q/tumour necrosis factor-related protein-9 (CTRP9) has been shown to be involved in diverse inflammatory diseases, we sought to assess the underlying impact of CTRP9 on NLRP3 inflammasome activation. In vitro, macrophages isolated from murine peritonea were stimulated with exogenous CTRP9, followed by lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP). We demonstrated that CTRP9 markedly augmented the activation of the NLRP3 inflammasome, as shown by increased mature IL-1ß secretion, triggering ASC speck formation and promoting pyroptosis. Mechanistically, CTRP9 increased the levels of NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). Suppressing ROS with N-acetylcysteine (NAC) or interfering with NOX2 by small interfering RNA weakened the promoting effect of CTRP9 on the NLRP3 inflammasome. Furthermore, NLRP3 inflammasome activation, pyroptosis and secretion of mature IL-1ß were significantly decreased in macrophages from CTRP9-KO mice compared to those from WT mice with the same treatment. In vivo, we established a sepsis model by intraperitoneal injection of LPS into WT and CTRP9-KO mice. CTRP9 knockout improved the survival rates of the septic mice and attenuated NLRP3 inflammasome-mediated inflammation. In conclusion, our study indicates that CTRP9 aggravates LPS-induced inflammation by promoting NLRP3 inflammasome activation via the NOX2/ROS pathway. CTRP9 could be a promising target for NLRP3 inflammasome-driven inflammatory diseases.

Progranulin mediates immune evasion of pancreatic ductal adenocarcinoma through regulation of MHCI expression.

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8+ T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC.

Exposing cryptic epitopes on the Venezuelan equine encephalitis virus E1 glycoprotein prior to treatment with alphavirus cross-reactive monoclonal antibody allows blockage of replication early in infection.

Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research.

Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1ß secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

Antibody Generation and Immunogenicity Analysis of EBV gp42 N-Terminal Region.

Epstein-Barr virus (EBV) is the first reported oncogenic virus and infects more than 90% of adults worldwide. EBV can establish a latent infection in B lymphocytes which is essential for persistence and transmission. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into a B cell. The N-terminal region of gp42 plays a key role in binding to gH/gL and triggering subsequent membrane fusion. However, no antibody has been reported to recognize this region and the immunogenicity of gp42 N-domain remains unknown. In the present study, we have generated a panel of nine mAbs against the gp42 N-terminal region (six mAbs to gp42-44-61aa and three mAbs to gp42-67-81aa). These mAbs show excellent binding activity and recognize different key residues locating on the gp42 N-domain. Among the nine mAbs, 4H7, 4H8 and 11G10 cross-react with rhLCV-gp42 while other mAbs specifically recognize EBV-gp42. Our newly obtained mAbs provide a useful tool for investigating the gp42 function and viral infection mechanism of γ-Herpesvirus. Furthermore, we assess the immunogenicity of the gp42 N-terminal region using the HBc149 particle as a carrier protein. The chimeric VLPs can induce high antibody titers and elicit neutralizing humoral responses to block EBV infection. More rational and effective designs are required to promote the gp42-N terminal region to become an epitope-based vaccine.

Tolerance Induced by Antigen-Loaded PLG Nanoparticles Affects the Phenotype and Trafficking of Transgenic CD4+ and CD8+ T Cells.

We have shown that PLG nanoparticles loaded with peptide antigen can reduce disease in animal models of autoimmunity and in a phase 1/2a clinical trial in celiac patients. Clarifying the mechanisms by which antigen-loaded nanoparticles establish tolerance is key to further adapting them to clinical use. The mechanisms underlying tolerance induction include the expansion of antigen-specific CD4+ regulatory T cells and sequestration of autoreactive cells in the spleen. In this study, we employed nanoparticles loaded with two model peptides, GP33-41 (a CD8 T cell epitope derived from lymphocytic choriomeningitis virus) and OVA323-339 (a CD4 T cell epitope derived from ovalbumin), to modulate the CD8+ and CD4+ T cells from two transgenic mouse strains, P14 and DO11.10, respectively. Firstly, it was found that the injection of P14 mice with particles bearing the MHC I-restricted GP33-41 peptide resulted in the expansion of CD8+ T cells with a regulatory cell phenotype. This correlated with reduced CD4+ T cell viability in ex vivo co-cultures. Secondly, both nanoparticle types were able to sequester transgenic T cells in secondary lymphoid tissue. Flow cytometric analyses showed a reduction in the surface expression of chemokine receptors. Such an effect was more prominently observed in the CD4+ cells rather than the CD8+ cells.

Development of a monoclonal antibody against PRRSV glycoprotein 3 using an immuodominant peptide as immunogen.

Glycoprotein 3 (GP3), a highly glycosylated membrane protein, is a protective antigen and minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV), and plays a crucial role in virus assembly and infection. In the present study, we synthesized 23 overlapping peptides span GP3 protein sequence and used pig anti-PRRSV serums to identify immunodominant peptides by indirect ELISA. Five immunodominant peptides GP3-P3, P4, P5, P6 and P7 were identified and GP3-P4 (P55LCPTRQAAAEILEPGKS72) was conjugated to carrier protein BSA. One mAb 1E5 against GP3 was generated from BALB/c mice immunized with the conjugates BSA-P4. The Characterization of mAb was identified by Western blot, Dot-ELISA, IPMA and IFA. We found that mAb 1E5 can specifically react with HP-PRRSV strains but not C-PRRSV or NADC30-like PRRSV strains tested in this study. Site-directed alanine substitution analysis revealed that 8 amino acid residues were involved in antibody binding, among them E65, L67 and P69 were critical residue recognized by mAb 1E5. Taken together, this study provided a novel strategy for generating specific mAbs against virus proteins by using immunodominant peptides as targets, and the mAb 1E5 may be useful for development of rapid differential detection method differentiating HP-PRRSV from C-PRRSV and NADC30-like PRRSV.

Human T cells engineered with a leukemia lipid-specific TCR enables donor-unrestricted recognition of CD1c-expressing leukemia.

Acute leukemia relapsing after chemotherapy plus allogeneic hematopoietic stem cell transplantation can be treated with donor-derived T cells, but this is hampered by the need for donor/recipient MHC-matching and often results in graft-versus-host disease, prompting the search for new donor-unrestricted strategies targeting malignant cells. Leukemia blasts express CD1c antigen-presenting molecules, which are identical in all individuals and expressed only by mature leukocytes, and are recognized by T cell clones specific for the CD1c-restricted leukemia-associated methyl-lysophosphatidic acid (mLPA) lipid antigen. Here, we show that human T cells engineered to express an mLPA-specific TCR, target diverse CD1c-expressing leukemia blasts in vitro and significantly delay the progression of three models of leukemia xenograft in NSG mice, an effect that is boosted by mLPA-cellular immunization. These results highlight a strategy to redirect T cells against leukemia via transfer of a lipid-specific TCR that could be used across MHC barriers with reduced risk of graft-versus-host disease.

Conserved immunogenic peptides of Ebola glycoprotein elicit immune response in human peripheral blood mononuclear cells.

In the past 45 years, ebolaviruses have periodically caused epidemics on the African continent. In December 2019, approval of a recombinant vector-based EBOV vaccine, named Ervebo, came as encouraging news; still, there is a long way to go in the development of an accessible, global, and pan-ebolavirus vaccine. The current study expanded our previous in silico work which was conducted on ebolavirus glycoprotein and this resulted in the identification of three potentially immunogenic peptides (P1 - FKRTSFFLWVIILFQRTFSIPL, P2 - LANETTQALQLF, and P3 - RATTELRTFSILNRKAIDF). An analysis to estimate the number of expected human leukocyte antigen (HLA) responders revealed that P1, P2, and P3 can potentially interact with 2540, 2150, and 2802 HLA alleles, respectively. Further, these peptides were subject to in vitro analysis wherein the human peripheral blood mononuclear cell proliferation and interferon-gamma (IFN-γ) production by peptide stimulated cells was studied in 10 healthy human blood samples with the help of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and a sandwich enzyme-linked immunosorbent assay (ELISA) respectively. P3 presented the best results, a significant (P < 0.05) peptide induced cell proliferation and IFN-γ stimulation for 8 and 10 samples, respectively, followed by P1 (5 and 6) and P2 (5 and 7). The in silico and in vitro results obtained in this study indicate the immunogenic potential of these peptides and warrant exploration of the effects on other cytokines as well as in vivo experimental validation.

Fel d1 Blocking Antibodies: A Novel Method to Reduce IgE-Mediated Allergy to Cats.

Fel d1 is an important allergen produced by cats that causes IgE reactions in up to 95% of cat-allergic adults. Immunotherapy to reduce human allergy to cats has demonstrated that people have the capacity to produce allergen-specific neutralizing antibodies that block IgE-mediated allergic responses. We wished to determine if "blocking" antibodies could be used to reduce the IgE binding ability of cat allergens prior to their exposure to humans. Here, we describe the characterization of Fel d1-specific antibodies. We demonstrated the efficacy of a rabbit polyclonal and an allergen-specific chicken IgY to bind to Fel d1 in cat saliva and block Fel d1-IgE binding and IgE-mediated basophil degranulation. Fel d1 blocking antibodies offer a new and exciting approach to the neutralization of cat allergens.

Expression, methylation and prognostic feature of EMILIN2 in Low-Grade-Glioma.

Low-grade gliomas (LGGs) are slow-growing brain cancer in central nervous system neoplasms. EMILIN2 is an extracellular matrix (ECM) protein which could influence the progress of some tumour which is unclear in LGG. In our study, the methylation, expression, prognosis and immune value of EMILIN2 in LGG were analysed through bioinformatics analysis. We analysed the LGG data from The Cancer Genome Atlas (TCGA) and discovered that the EMILIN2 expression, negatively correlated to the EMILIN2 methylation, could predict a poor prognosis and was associated with different clinical parameters. Moreover, univariate and multivariate Cox regression were performed in CGGA, which showed that the EMILIN2 could be an independent prognostic biomarker in LGG. Moreover, EMILIN2 expression showed a correlation with gene makers in some immune cells, which identified the significance of EMILIN2 in immune infiltration. Finally, we used RT-PCR to verify the EMILIN2 expression level in different grades which showed there were significantly different (P < 0.05). Similarly, high expression of EMILIN2 could predict a poor prognosis (P = 0.0078). In conclusion, EMILIN2 could act as an independent prognostic biomarker which might be associated with the malignancy and development of gliomas and play a crucial role in glioma in immune infiltration.

Toward an understanding of tissue factor pathway inhibitor-2 as a novel serodiagnostic marker for clear cell carcinoma of the ovary.

AIMS: Tissue factor pathway inhibitor (TFPI)-2 has recently emerged as a serodiagnostic marker for patients with epithelial ovarian cancer (EOC), especially clear cell carcinoma (CCC). This review discusses the biological properties of TFPI-2 and why serum levels are elevated in CCC patients. METHODS: A comprehensive literature search was conducted in PubMed up until March, 2021. RESULTS: TFPI-2 is a Kunitz-type protease inhibitor and negatively regulates the enzymatic activities, such as plasmin. TFPI-2 has been characterized as a tumor suppressor gene and was frequently downregulated through promoter hypermethylation in various human cancers. In contrast, TFPI-2 was overexpressed only in CCC. TFPI-2 may be involved in the pathophysiology of CCC, possibly through regulation of coagulation system, stabilization of extracellular matrix (ECM), and induction of intracellular signal transduction. TFPI-2 suppresses tissue factor-induced hypercoagulation in a hypoxic environment. TFPI-2, secreted by CCC cells, platelets, and adjacent vascular endothelial cells, may suppress tumor growth and invasion through ECM remodeling. Nuclear TFPI-2 may suppress matrix metalloproteinase production via transcription factors and modulate caspase-mediated cell apoptosis. CCC cells may upregulate the TFPI-2 expression to adapt to survival in the demanding environment. TFPI-2 is secreted by CCC cells and enters the systemic circulation, resulting in elevated blood levels. DISCUSSION: Serum TFPI-2 reflects the overexpression of TFPI-2 in CCC tissues and is a potential serodiagnostic marker. Further research is needed to explore the expression, clinical significance, biological function, and potential mechanism of TFPI-2 in CCC.

Generation of triacyl lipopeptide-modified glycoproteins by metabolic glycoengineering as the neoantigen to boost anti-tumor immune response.

The lack of tumor specific antigens (TSA) and the immune tolerance are two major obstacles for the immunotherapy of cancer. Current immune checkpoint inhibitors (ICIs) show clinical responses in only limited subsets of cancer patients, which, to some extent, depends on the mutation load of tumor cells that may generate neoantigens. Here, we aimed to generate a neoantigen MDP to exhibit stronger anti-tumor efficacy. Methods: In this study, we utilized chemically modified sialic acid precursor tetra acetyl-N-azidoacetyl-mannosamine (AC4ManNAZ) to engineer the glycoproteins on the membranes of tumor cells for the covalent ligation of hapten adjuvant Pam3CSK4 in vivo, which eventually generated a neoantigen, i.e., ManNAZ-DBCO-Pam3CSK4 (MDP), on tumor cells. The high labeling efficiency, relatively specific biodistribution in tumor tissues and the anti-tumor efficacy were confirmed in the syngeneic murine models of the breast cancer and the lung cancer. Results: The generation of MDP neoantigen in tumor-bearing mice significantly evoked both the humoral and the T-cell-dependent antitumor immune responses, resulting in a strong inhibition on the growth of the breast cancer and the lung cancer allografts and significantly prolonged survival of tumor-bearing mice. Interestingly, MDP neoantigen was able to dramatically increase the sensitivity of cancer cells to ICIs and greatly enhance the anti-tumor efficacy in the murine models of both breast cancer and the lung cancer, which showed no or low responses to the immunotherapy with anti-PD1 antibody alone. Conclusions: We developed a simple metabolic glycoengineering method to artificially generate neoantigens on tumor cells to enhance tumor cell immunogenicity, which is able to significantly improve the response and the clinical outcome of ICIs.

Harnessing EV communication to restore antitumor immunity.

Restoring effective anti-tumor immune responses to cure cancer is a promising strategy, but challenging to achieve due to the intricate crosstalk between tumor and immune cells. While it is established that tumor cells acquire traits to escape immune recognition, the involvement of extracellular vesicles (EVs) in curbing immune cell activation is rapidly emerging. By assisting cancer cells in spreading immunomodulatory signals in the form of (glyco)proteins, lipids, nucleic acids and metabolic regulators, EVs recently emerged as versatile mediators of immune suppression. Blocking their action might reactivate immune cell function and natural antitumor immune responses. Alternatively, EV communication may be exploited to boost anti-tumor immunity. Indeed, novel insights into EV biology paved the way for efficient ex vivo production of 'rationally engineered' EVs that function as potent antitumor vaccines or carry out specific functional tasks. In this review we discuss the latest findings on immune regulation by cancer EVs and explore how EV-mediated communication can be either targeted or harnessed to restore immunity as a means for cancer therapy.

Baseline serum B-cell maturation antigen levels predict time to disease progression for patients with smoldering multiple myeloma.

Multiple myeloma (MM) patients with smoldering (S) disease are defined by a lack of CRAB/SLiM criteria but may transform into disease requiring treatment. The International Myeloma Working Group risk stratification model for SMM uses serum M-protein, serum-free light chain ratio, and bone marrow plasma cell percentage. We investigated whether baseline serum B-cell maturation antigen (sBCMA) levels are predictive of disease progression among 65 patients with SMM. A receiver operating characteristic curve was used to establish a definition for high-risk baseline sBCMA. Mantel Byar analysis was used to examine whether high-risk sBCMA was correlated with shorter time to transformation, and a time-dependent cox proportional hazard was used to determine whether it is independent of other risk factors. A z test for proportions was used to compare the percentage of patients that progressed among high-risk versus low-risk sBCMA patients. A baseline sBCMA level ≥137.5 mg/ml was found to be the optimal cutoff between high- and low-risk SMM patients. Patients with high-risk sBCMA levels had a shorter time to transformation (P = .000332). sBCMA was also higher at the time of transformation than baseline levels (P = .0116). sBCMA was the only variable found to be significantly predictive of time to transformation and additionally was found to be independent of other risk factors. In this study, we have shown for the first time that sBCMA levels predict transformation of SMM to active disease and that these levels increase at the time of transformation. These results are consistent with other studies showing that active MM patients undergoing therapy with higher baseline sBCMA levels are more likely to progress early and its levels increase at the time of disease progression.

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins.

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.