Immune checkpoint blockade therapy has demonstrated significant therapeutic efficacy in certain cancer types; however, the impact of dietary restriction remains scarcely reported in this context. This study aimed to investigate the influence of dietary restriction on anti-PDL-1 therapy and the interplay of immune cells within this context. Using an anti-PDL-1 regimen combined with dietary restrictions, tumor progression was assessed in LLC-bearing mice. Flow cytometry was employed to analyze immune cell infiltration and differentiation levels within the tumor microenvironment. The expression of mTORC1/B7-H3 in tumors subjected to dietary restriction was also examined. LLC tumors with elevated B7-H3 expression were validated in mice to determine its inhibitory effect on immune cell proliferation and differentiation. A CD3/B7-H3 chimeric antibody was developed for therapeutic intervention in B7-H3 overexpressing tumors, with subsequent T cell responses assessed through flow cytometry. Dietary restriction potentiated the effect of anti-PDL1 therapy by suppressing the intratumorally mTORC1/B7-H3 axis. In vivo experiments demonstrated that elevated B7-H3 expression in tumors reduced infiltration and activation of CD8 + T cells within the tumor, while it did not affect tumor-infiltrating Tregs. In vitro studies revealed that high B7-H3 expression influenced the proliferation and activation of CD8 + T cells within a Coculture system. The constructed CD3/B7-H3 chimeric antibody prominently activated TCR within B7-H3 overexpressing tumors and impeded tumor progression. The findings suggest that dietary restriction enhances the efficacy of immune checkpoint blockade by modulating the intratumoral mTORC1/B7-H3 axis.

Chordoma is a rare bone tumor that frequently recurs after surgery, and the prognosis is poor with current treatments. This study aimed to identify potential novel immunotherapeutic targets for chordomas by identifying target proteins in clinical samples as well as tumor microenvironmental factors to enhance efficacy. Fourteen chordoma samples were analyzed by single-cell RNA sequencing, and B7-H3 and IL-7 were identified as potential targets and potentiators, respectively. B7-H3-targeted chimeric antigen receptor T (CAR-T) cells and B7-H3 CAR-T cells expressing IL-7 were synthesized and their anti-tumor activity evaluated in vitro, including in primary chordoma organoid models. The B7-H3 CAR-T/IL-7 therapy showed enhanced cytotoxicity and prolonged duration of action against tumor cells. Additionally, IL-7 modulated favorable subpopulations of cultured CAR-T cells, diminished immune checkpoint expression on T-cell surfaces, and enhanced T-cell functionality. The incorporation of IL-7 molecules into the B7-H3 CAR structure augmented CAR-T-cell function and improved CAR-T-cell efficacy, thus providing a novel dual therapeutic strategy for chordoma treatment.

In recent years, in the development of emerging immunotherapy, B7-H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)-T target against glioma and other tumours, and aroused extensive attention. However, B7-H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7-H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7-H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5-methylcytosine reader Y-box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7-H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy.

Chimeric antigen receptor (CAR) T-cells targeting Fibroblast Growth Factor Receptor 4 (FGFR4), a highly expressed surface tyrosine receptor in rhabdomyosarcoma (RMS), are already in the clinical phase of development, but tumour heterogeneity and suboptimal activation might hamper their potency. Here we report an optimization strategy of the co-stimulatory and targeting properties of a FGFR4 CAR. We replace the CD8 hinge and transmembrane domain and the 4-1BB co-stimulatory domain with those of CD28. The resulting CARs display enhanced anti-tumor activity in several RMS xenograft models except for an aggressive tumour cell line, RMS559. By searching for a direct target of the RMS core-regulatory transcription factor MYOD1, we identify another surface protein, CD276, as a potential target. Bicistronic CARs (BiCisCAR) targeting both FGFR4 and CD276, containing two distinct co-stimulatory domains, have superior prolonged persistent and invigorated anti-tumor activities compared to the optimized FGFR4-specific CAR and the other BiCisCAR with the same 4-1BB co-stimulatory domain. Our study thus lays down the proof-of-principle for a CAR T-cell therapy targeting both FGFR4 and CD276 in RMS.

BACKGROUND: Advances in pediatric oncology have occurred for some cancers; however, new therapies for sarcoma have been inadequate. Cellular immunotherapy using chimeric antigen receptor (CAR) T cells has shown dramatic benefits in leukemia, lymphoma, and multiple myeloma but has been far less successful in pediatric solid tumors such as rhabdomyosarcoma (RMS) and osteosarcoma (OS). Balancing issues of \"on-target, off-tumor toxicity\", investigators have identified B7-H3 as a broadly expressed tumor antigen with otherwise restricted expression on normal tissues. We hypothesized that rapid homing via a chemokine receptor and CAR engagement through B7-H3 would enhance CAR T cell efficacy in solid tumors.
METHODS: We generated B7-H3 CAR T cells that also express the Interleukin-8 (IL-8) receptor, CXCR2. Cytokine production, flow cytometry, Seahorse assays and RNA sequencing were used to compare the B7-H3 CXCR2 (BC2) CAR T cells with B7-H3 CAR T cells. We developed an IL-8 overexpressing human RMS mouse model to test homing and cytotoxicity in vivo.
RESULTS: We demonstrate that IL-8 is expressed by RMS and OS and expression significantly increases after radiation. Overexpression of an IL-8 receptor, CXCR2, on B7-H3 CAR T cells enhances homing into IL-8 expressing tumors, augments T cell metabolism and leads to significant tumor regression.
CONCLUSIONS: These findings warrant further investigation into the use of BC2 CAR T cells as a treatment for patients with RMS, OS and other B7-H3-expressing, IL-8 producing solid tumors.

UNASSIGNED: Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes.
UNASSIGNED: A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification.
UNASSIGNED: NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56- cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80+ cytotoxic T-cells at the more advanced maturation stages, mostly in adults.
UNASSIGNED: Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80- phenotype.

OBJECTIVE: B7 molecules (B7s) are crucial synergistic signals for effective immune surveillance against tumor cells. While previous studies have explored the association between the B7 family and cancer, most have been limited to specific genes or cancer subtypes.
METHODS: Our study utilized multi-omics data to investigate potential correlations between B7s expression (B7s exp.) and prognosis, clinicopathological features, somatic mutations (SMs), copy number variations (CNVs), immune characteristics, tumor microenvironment (TME), microsatellite instability, tumor mutation burden, immune checkpoint gene (ICG), and drug responsiveness in TCGA tumors. Furthermore, the connection between B7s exp. and immunotherapy (IT) performance assessed in various validated datasets. Following this, immune infiltration analysis (IIA) was conducted based on B7s exp., CNVs, or SMs in bladder cancer (BLCA), complemented by real-time PCR (RT-PCR) and protein confirmation of B7-H3.
RESULTS: Across most cancer types, B7s exp. was related to prognosis, clinicopathological characteristics, mutations, CNVs, ICG, TMB, TME. The examination of sensitivity to anticancer drugs unveiled correlations between B7 molecules and different drug sensitivities. Specific B7s exp. patterns were linked to the clinical effectiveness of IT. Using GSEA, several enriched immune-related functions and pathways were identified. Particularly in BLCA, IIA revealed significant connections between B7 CNVs, mutation status, and various immune cell infiltrates. RT-PCR confirmed elevated B7-H3 gene levels in BLCA tumor tissues.
CONCLUSIONS: This study confirmed the significance of B7s exp. and genomic changes in predicting outcomes and treatment across different cancer types. Moreover, they indicate a critical function of B7s in BLCA and their potential as IT biomarkers.

Despite the success of immunotherapy, overcoming immunoresistance in cancer remains challenging. We identified a unique niche of tumor-associated macrophages (TAMs), coexpressing T cell immunoglobulin and mucin domain-containing 3 (TIM3) and V-domain immunoglobulin suppressor of T cell activation (VISTA), that dominated human and mouse tumors resistant to most of the currently used immunotherapies. TIM3+VISTA+ TAMs were sustained by IL-4-enriching tumors with low (neo)antigenic and T cell-depleted features. TIM3+VISTA+ TAMs showed an anti-inflammatory and protumorigenic phenotype coupled with inability to sense type I interferon (IFN). This was established with cancer cells succumbing to immunogenic cell death (ICD). Dying cancer cells not only triggered autocrine type I IFNs but also exposed HMGB1/VISTA that engaged TIM3/VISTA on TAMs to suppress paracrine IFN-responses. Accordingly, TIM3/VISTA blockade synergized with paclitaxel, an ICD-inducing chemotherapy, to repolarize TIM3+VISTA+ TAMs to proinflammatory TAMs that killed cancer cells via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. We propose targeting TIM3+VISTA+ TAMs to overcome immunoresistant tumors.

Although inhibitors targeting the PD1/PD-L1 immune checkpoint are showing comparably good outcomes, a significant percentage of head and neck squamous cell carcinoma (HNSCC) patients do not respond to treatment. Apart from using different treatment strategies, another possibility would be to target other immune checkpoints operating in these non-responding tumors. To obtain an overview of which checkpoint ligands are expressed on HNSCC tumor cells and if these ligands are affected by HGF/MET signaling, we used mRNA sequencing and antibody-based techniques for identifying checkpoint ligands in six HNSCC tumor cell lines. Furthermore, we compared our results to mRNA sequencing data. From the checkpoint ligands we investigated, VISTA was expressed the highest at the RNA level and was also the most ubiquitously expressed. PD-L2 and B7-H3 were expressed comparably lower and were not present in all cell lines to the same extent. B7-H4, however, was only detectable in the Detroit 562 cell line. Concerning the effect of HGF on the ligand levels, PD-L2 expression was enhanced with HGF stimulation, whereas other checkpoint ligand levels decreased with stimulation. B7-H4 levels in the Detroit 562 cell line drastically decreased with HGF stimulation. This is of interest because both the checkpoint ligand and the growth factor are reported to be connected to epithelial-mesenchymal transition in the literature.

Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment but has shown limited efficacy in gynecologic cancers. VISTA (V-domain Ig suppressor of T-cell activation), a member of the B7 family, is emerging as another checkpoint that regulates the anti-tumor immune responses within the tumor microenvironment. This paper reviews the structure, expression, and mechanism of action of VISTA. Furthermore, it highlights recent advances in VISTA-blocking therapies and their potential in improving outcomes for patients with gynecologic cancers. By understanding the role of VISTA in mediating the immune evasion of gynecologic tumors, we can develop more effective combinatory treatment strategies that could overcome resistance to current ICB therapies.