High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-ß7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-ß7, LAG3, TGFß/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-ß7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-ß7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Comprehensive analysis and immunohistochemistry localization of NRP1 expression in pancancer and normal individual tissues in relation to SARS­CoV­2 susceptibility.

Neuropilin 1 (NRP1/CD304) is a typical membrane-bound co-receptor for vascular endothelial growth factor, semaphorin family members and viral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, NRP1 expression levels across cancer types and the potential role of SARS-CoV-2 infection in patients with cancer are not clear. Online databases, such as The Cancer Genome Atlas database of Human Protein Atlas, Gene Expression Profiling Interactive Analysis and cBioPortal were used for the expression analysis in this study. Immunohistochemical (IHC) staining for NRP1 was performed in the tissues of patients with non-small cell carcinoma. As a result, it was found that NRP1 mRNA and protein expression levels were highest in the female reproductive tissues and the respiratory system, specifically in the nasopharynx, bronchus and fallopian tube, as well as in adipocytes, hepatic stellate cells, Sertoli cells, endothelial cells and dendritic cells. IHC showed that the NRP1 protein was mainly localized to the cytoplasm and membrane in the tissues of patients with non-small cell carcinoma, demonstrating its role in lung infection by SARS-CoV-2, due to invasion of cell membranes by the virus. Levels of NRP1 mRNA were significantly increased in lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma (KIRC), pancreatic adenocarcinoma, stomach adenocarcinoma and thymoma, and significantly decreased in cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney chromophobe, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma, compared with corresponding healthy tissues in pancancer, indicating roles for viral invasion in most cancer types. Moreover, low NRP1 expression was significantly associated with long overall survival (OS) time in adrenocortical carcinoma, brain lower grade glioma, stomach adenocarcinoma and uveal melanoma, but with short OS time in KIRC only. The ENST00000374867.6 (NRP1-202) isoform is most highly expressed in most cancer types and thus could be involved in tumorigenesis and SARS-CoV-2 invasion in cancer patients. NRP1 may be involved in SARS-CoV-2 invasion in patients with cancer, including those with lung cancer.

Evaluation of diagnostic efficacy of NRP-1/CD304 in hematological diseases.

BACKGROUND: Previous studies had explored the diagnostic or prognostic value of NRP-1/CD304 in blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), and B-cell acute lymphoblastic leukemia (B-ALL), whereas the expression and application value of NRP-1/CD304 in other common hematological diseases have not been reported. METHODS: Bone marrow samples from 297 newly diagnosed patients with various hematological diseases were collected to detect the expression of NRP-1/CD304 by flow cytometry (FCM). The diagnostic efficacy of NRP-1/ CD304-positive diseases was analyzed by receiver operating characteristic (ROC) curve, and the area under the ROC curve (AUC) was compared. RESULTS: In the research cohort, the total positive rate of NRP-1/CD304 was 14.81% (44/297), mainly distributed in BPDCN (100%, 6/6), B-ALL (48.61%, 35/72), and AML (4.48%, 3/67), with statistically significant differences (p < 0.01). Other diseases, such as T-cell acute lymphoblastic leukemia (T-ALL), B-cell non-Hodgkin lymphoma (B-NHL), T/NK-cell lymphoma and plasma cell neoplasms, did not express NRP-1/CD304. The AUC of NRP-1/CD304 was 0.936 (95% CI 0.898-0.973), 0.723 (95% CI 0.646-0.801), and 0.435 (95% CI 0.435) in BPDCN, B-ALL and AML, respectively. Besides, CD304 was commonly expressed in B-ALL with BCR-ABL1 gene rearrangement (p = 0.000), and CD304 expression was positively correlated with CD34 co-expression (p = 0.009) and CD10 co-expression (p = 0.007). CONCLUSIONS: NRP-1/CD304 is only expressed in BPDCN, B-ALL and AML, but not in other common hematological diseases. This indicates that NRP-1/CD304 has no obvious diagnostic and follow-up study value in hematological diseases other than BPDCN, B-ALL, and AML.

Natural Product Cordycepin (CD) Inhibition for NRP1/CD304 Expression and Possibly SARS-CoV-2 Susceptibility Prevention on Cancers.

NRP1/CD304 is a typical membrane-bound co-receptor for the vascular endothelial cell growth factor (VEGF), semaphorin family members, and viral SARS-CoV-2. Cordycepin (CD) is a natural product or active gradient from traditional Chinese medicine (TCM) from Cordyceps militaris Link and Ophiocordyceps sinensis (Berk.). However, NRP1 expression regulation via CD in cancers and the potential roles and mechanisms of SARS-CoV-2 infection are not clear. In this study, online databases were analyzed, Western blotting and quantitative RT-PCR were used for NRP1 expression change via CD, molecular docking was used for NRP/CD interaction, and a syncytial formation assay was used for CD inhibition using a pseudovirus SARS-CoV-2 entry. As a result, we revealed that CD inhibits NRP1 expressed in cancer cells and prevents viral syncytial formation in 293T-hACE2 cells, implying the therapeutic potential for both anti-cancer and anti-viruses, including anti-SARS-CoV-2. We further found significant associations between NRP1 expressions and the tumor-immune response in immune lymphocytes, chemokines, receptors, immunostimulators, immune inhibitors, and major histocompatibility complexes in most cancer types, implying NRP1's roles in both anti-cancer and anti-SARS-CoV-2 entry likely via immunotherapy. Importantly, CD also downregulated the expression of NRP1 from lymphocytes in mice and downregulated the expression of A2AR from the lung cancer cell line H1975 when treated with CD, implying the NRP1 mechanism probably through immuno-response pathways. Thus, CD may be a therapeutic component for anti-cancer and anti-viral diseases, including COVID-19, by targeting NRP1 at least.

Expression of CD304/neuropilin-1 in adult b-cell lymphoblastic leukemia/lymphoma and its utility for the measurable residual disease assessment.

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.

miR-24 Targets the Transmembrane Glycoprotein Neuropilin-1 in Human Brain Microvascular Endothelial Cells.

Neuropilin-1 is a transmembrane glycoprotein that has been implicated in several processes including angiogenesis and immunity. Recent evidence has also shown that it is implied in the cellular internalization of the severe acute respiratory syndrome coronavirus (SARS-CoV-2), which causes the coronavirus disease 2019 (COVID-19). We hypothesized that specific microRNAs can target Neuropilin-1. By combining bioinformatic and functional approaches, we identified miR-24 as a regulator of Neuropilin-1 transcription. Since Neuropilin-1 has been shown to play a key role in the endothelium-mediated regulation of the blood-brain barrier, we validated miR-24 as a functional modulator of Neuropilin-1 in human brain microvascular endothelial cells (hBMECs), which are the most suitable cell line for an in vitro blood-brain barrier model.

CD304/neuropilin-1 is a very useful and dependable marker for the measurable residual disease assessment of B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Measurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC-based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin-1, in the assessment of MFC-based MRD. METHODS: Expression patterns of CD304 were studied in leukemic blasts from BCP-ALL patients and in normal precursor B cells (NPBC) from uninvolved non-BCP-ALL bone marrow samples using 10-color MFC. MRD was monitored at end-of-induction (EOI; Days 35-40) and end-of-consolidation (Day 78-80) time points. RESULTS: We studied CD304 expression in 300 pediatric BCP-ALL patients and found it positive in BCP-ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6-RUNX1 (p < .001) as well as BCR-ABL1 (p = .019) and inversely associated with TCF3-PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI-MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low-level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI-MRD samples. CONCLUSIONS: CD304 is commonly expressed in leukemic blasts of BCP-ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC-based MRD assay in BCP-ALL.

Differential expression of CD73, CD86 and CD304 in normal vs. leukemic B-cell precursors and their utility as stable minimal residual disease markers in childhood B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Optimal discrimination between leukemic blasts and normal B-cell precursors (BCP) is critical for treatment monitoring in BCP acute lymphoblastic leukemia (ALL); thus identification of markers differentially expressed on normal BCP and leukemic blasts is required. METHODS: Multicenter analysis of CD73, CD86 and CD304 expression levels was performed in 282 pediatric BCP-ALL patients vs. normal bone marrow BCP, using normalized median fluorescence intensity (nMFI) values. RESULTS: CD73 was expressed at abnormally higher levels (vs. pooled normal BCP) at diagnosis in 71/108 BCP-ALL patients (66%), whereas CD304 and CD86 in 119/202 (59%) and 58/100 (58%) patients, respectively. Expression of CD304 was detected at similar percentages in common-ALL and pre-B-ALL, while found at significantly lower frequencies in pro-B-ALL. A significant association (p = 0.009) was found between CD304 expression and the presence of the ETV6-RUNX1 fusion gene. In contrast, CD304 showed an inverse association with MLL gene rearrangements (p = 0.01). The expression levels of CD73, CD86 and CD304 at day 15 after starting therapy (MRD15) were stable or higher than at diagnosis in 35/37 (95%), 40/56 (71%) and 19/41 (46%) cases investigated, respectively. This was also associated with an increased mean nMFI at MRD15 vs. diagnosis of +24 and +3 nMFI units for CD73 and CD86, respectively. In addition, gain of expression of CD73 and CD86 at MRD15 for cases that were originally negative for these markers at diagnosis was observed in 16% and 18% of cases, respectively. Of note, CD304 remained aberrantly positive in 63% of patients, despite its levels of expression decreased at follow-up in 54% of cases. CONCLUSIONS: Here we show that CD73, CD86 and CD304 are aberrantly (over)expressed in a substantial percentage of BCP-ALL patients and that their expression profile remains relatively stable early after starting therapy, supporting their potential contribution to improved MRD analysis by flow cytometry.