Background Immunotherapy against solid tumors has long been hampered by the development of immunosuppressive tumor microenvironment, and the lack of a specific tumor-associated antigen that could be targeted in different kinds of solid tumors. Human leukocyte antigen G (HLA-G) is an immune checkpoint protein (ICP) that is neoexpressed in most tumor cells as a way to evade immune attack and has been recently demonstrated as a useful target for chimeric antigen receptor (CAR)-T therapy of leukemia by in vitro studies. Here, we design and test for targeting HLA-G in solid tumors using a CAR strategy. Methods We developed a novel CAR strategy using natural killer (NK) cell as effector cells, featuring enhanced cytolytic effect via DAP12-based intracellular signal amplification. A single-chain variable fragment (scFv) against HLA-G is designed as the targeting moiety, and the construct is tested both in vitro and in vivo on four different solid tumor models. We also evaluated the synergy of this anti-HLA-G CAR-NK strategy with low-dose chemotherapy as combination therapy. Results HLA-G CAR-transduced NK cells present effective cytolysis of breast, brain, pancreatic, and ovarian cancer cells in vitro, as well as reduced xenograft tumor growth with extended median survival in orthotopic mouse models. In tumor coculture assays, the anti-HLA-G scFv moiety promotes Syk/Zap70 activation of NK cells, suggesting reversal of the HLA-G-mediated immunosuppression and hence restoration of native NK cytolytic functions. Tumor expression of HLA-G can be further induced using low-dose chemotherapy, which when combined with anti-HLA-G CAR-NK results in extensive tumor ablation both in vitro and in vivo. This upregulation of tumor HLA-G involves inhibition of DNMT1 and demethylation of transporter associated with antigen processing 1 promoter. Conclusions Our novel CAR-NK strategy exploits the dual nature of HLA-G as both a tumor-associated neoantigen and an ICP to counteract tumor spread. Further ablation of tumors can be boosted when combined with administration of chemotherapeutic agents in clinical use. The readiness of this novel strategy envisions a wide applicability in treating solid tumors.

Background: The tumor microenvironment (TME) plays a crucial role in virtually every aspect of tumorigenesis of glioblastoma multiforme (GBM). A dysfunctional TME promotes drug resistance, disease recurrence, and distant metastasis. Recent evidence indicates that exosomes released by stromal cells within the TME may promote oncogenic phenotypes via transferring signaling molecules such as cytokines, proteins, and microRNAs. Results: In this study, clinical GBM samples were collected and analyzed. We found that GBM-associated macrophages (GAMs) secreted exosomes which were enriched with oncomiR-21. Coculture of GAMs (and GAM-derived exosomes) and GBM cell lines increased GBM cells’ resistance against temozolomide (TMZ) by upregulating the prosurvival gene programmed cell death protein 4 (PDCD4) and stemness markers SRY (sex determining region y)-box 2 (Sox2), signal transducer and activator of transcription 3 (STAT3), Nestin, and miR-21-5p and increasing the M2 cytokines interleukin 6 (IL-6) and transforming growth factor beta 1(TGF-β1) secreted by GBM cells, promoting the M2 polarization of GAMs. Subsequently, pacritinib treatment suppressed GBM tumorigenesis and stemness; more importantly, pacritinib-treated GBM cells showed a markedly reduced ability to secret M2 cytokines and reduced miR-21-enriched exosomes secreted by GAMs. Pacritinib-mediated effects were accompanied by a reduction of oncomiR miR-21-5p, by which the tumor suppressor PDCD4 was targeted. We subsequently established patient-derived xenograft (PDX) models where mice bore patient GBM and GAMs. Treatment with pacritinib and the combination of pacritinib and TMZ appeared to significantly reduce the tumorigenesis of GBM/GAM PDX mice as well as overcome TMZ resistance and M2 polarization of GAMs. Conclusion: In summation, we showed the potential of pacritinib alone or in combination with TMZ to suppress GBM tumorigenesis via modulating STAT3/miR-21/PDCD4 signaling. Further investigations are warranted for adopting pacritinib for the treatment of TMZ-resistant GBM in clinical settings.

HLA‐G is considered as an immune checkpoint protein and a tumor‐associated antigen. In the previous work, it is reported that CAR‐NK targeting of HLA‐G can be used to treat certain solid tumors. However, the frequent co‐expression of PD‐L1 and HLA‐G) and up‐regulation of PD‐L1 after adoptive immunotherapy may decrease the effectiveness of HLA‐G‐CAR. Therefore, simultaneous targeting of HLA‐G and PD‐L1 by multi‐specific CAR could represent an appropriate solution. Furthermore, gamma‐delta T (γδT) cells exhibit MHC‐independent cytotoxicity against tumor cells and possess allogeneic potential. The utilization of nanobodies offers flexibility for CAR engineering and the ability to recognize novel epitopes. In this study, Vδ2 γδT cells are used as effector cells and electroporated with an mRNA‐driven, nanobody‐based HLA‐G‐CAR with a secreted PD‐L1/CD3ε Bispecific T‐cell engager (BiTE) construct (Nb‐CAR.BiTE). Both in vivo and in vitro experiments reveal that the Nb‐CAR.BiTE‐γδT cells could effectively eliminate PD‐L1 and/or HLA‐G‐positive solid tumors. The secreted PD‐L1/CD3ε Nb‐BiTE can not only redirect Nb‐CAR‐γδT but also recruit un‐transduced bystander T cells against tumor cells expressing PD‐L1, thereby enhancing the activity of Nb‐CAR‐γδT therapy. Furthermore, evidence is provided that Nb‐CAR.BiTE redirectes γδT into tumor‐implanted tissues and that the secreted Nb‐BiTE is restricted to the tumor site without apparent toxicity.

Background: Glioblastoma (GBM) is the most common and lethal primary malignant glioma in adults. Dendritic cell (DC) vaccines have demonstrated promising results in GBM clinical trials. However, some patients do not respond well to DC therapy, with survival rates similar to those of conventional therapy. We retrospectively analyzed clinical and laboratory data to evaluate the factors affecting vaccine treatment. Methods: Forty-seven patients with de novo GBM were enrolled at China Medical University Hospital between 2005 and 2010 and divided into two subgroups. One subgroup of 27 patients received postsurgical adjuvant immunotherapy with autologous dendritic cell/tumor antigen vaccine (ADCTA) in conjunction with conventional treatment of concomitant chemoradiotherapy (CCRT) with temozolomide. The other 20 patients received only postsurgical conventional treatment without immunotherapy. Immunohistochemistry for CD45, CD4, CD8, programed death ligand 1 (PD-L1), and programed death 1 (PD-1) was performed on sections of surgical tumor specimens and peripheral blood mononuclear cells (PBMCs). Pearson’s correlation, Cox proportional hazard model, and Kaplan–Meier analyses were performed to examine the correlations between the prognostic factors and survival rates. Results: Younger age (<57 years), gross total resection, and CCRT and PD-1⁺ lymphocyte counts were significant prognostic factors of overall survival (OS) and progression-free survival (PFS) in the ADCTA group. Sex, CD45⁺ lymphocyte count, CD4⁺ or CD8⁺ lymphocyte count, tumor PD-L1 expression, isocitrate dehydrogenase 1 mutation, and O6 methylguanine-DNA methyltransferase promoter methylation status were not significant factors in both groups. In the ADCTA group, patients with tumor-infiltrating lymphocytes (TILs) with a lower PD-1⁺/CD8⁺ ratio (≤0.21) had longer OS and PFS (median OS 60.97 months, P < 0.001 and PFS 11.2 months, P < 0.008) compared to those with higher PD-1⁺/CD8⁺ ratio (>0.21) (median OS 20.07 months, P < 0.001 and PFS 4.43 months, P < 0.008). Similar results were observed in patients’ PBMCs; lymphocyte counts with lower PD-1⁺/CD8⁺ ratio (≤0.197) had longer OS and PFS. There was a significant correlation of PD-1⁺/CD8⁺ ratio between TILs and PBMCs (Pearson’s correlation R² = 0.6002, P < 0.001). By contrast, CD4⁻, CD8⁻, but PD-1⁺, CD45⁺ tumor-infiltrating lymphocytes have no impact on OS and PFS (P = 0.073 and P = 0.249, respectively). Conclusion: For patients receiving DC vaccine adjuvant therapy, better outcomes are predicted in patients with younger age, with TILs or PBMCs with lower PD-1⁺/CD8⁺ ratio, with gross tumor resection, and receiving CCRT.

Bacteremia is a life-threatening complication of infectious diseases. Bacteremia can be predicted using machine learning (ML) models, but these models have not utilized cell population data (CPD). The derivation cohort from emergency department (ED) of China Medical University Hospital (CMUH) was used to develop the model and was prospectively validated in the same hospital. External validation was performed using cohorts from ED of Wei-Gong Memorial Hospital (WMH) and Tainan Municipal An-Nan Hospital (ANH). Adult patients who underwent complete blood count (CBC), differential count (DC), and blood culture tests were enrolled in the present study. The ML model was developed using CBC, DC, and CPD to predict bacteremia from positive blood cultures obtained within 4 h before or after the acquisition of CBC/DC blood samples. This study included 20,636 patients from CMUH, 664 from WMH, and 1622 patients from ANH. Another 3143 patients were included in the prospective validation cohort of CMUH. The CatBoost model achieved an area under the receiver operating characteristic curve of 0.844 in the derivation cross-validation, 0.812 in the prospective validation, 0.844 in the WMH external validation, and 0.847 in the ANH external validation. The most valuable predictors of bacteremia in the CatBoost model were the mean conductivity of lymphocytes, nucleated red blood cell count, mean conductivity of monocytes, and neutrophil-to-lymphocyte ratio. ML model that incorporated CBC, DC, and CPD showed excellent performance in predicting bacteremia among adult patients with suspected bacterial infections and blood culture sampling in emergency departments.