Subclone from CT26 resistant to anti-PD-1 therapy associated with increased expression of genes related to glucocorticoids.

BACKGROUND: Although the use of anti-PD-1 antibodies has fundamentally changed traditional cancer treatment, most patients are resistant to anti-PD-1 treatment. Glucocorticoids (GCs) play an important role in tumorigenesis and tumor progression, but the role of endogenous GCs in resistance to anti-PD-1 antibody therapy remains unclear. METHODS: Single cell-derived cell lines (SCDCLs) were generated from a colorectal cancer cell line (CT26) using limiting dilution. We analyzed tumor tissues from anti-PD-1 antibody-treated and untreated mice inoculated with SCDCLs via transcriptome sequencing and flow cytometry to detect pathway activity and immune cell composition changes in the tumor microenvironment. RESULTS: Five SCDCLs were inoculated into wild-type BALB/c mice (all tumorigenic). Single-cell clone (SCC)-2 exhibited the slowest growth rates both in vivo and in vitro compared to other single-cell clones, and better long-term survival than SCC1 and CT26. Flow cytometry showed that SCC2 tumor-bearing mice exhibited significantly higher infiltration of T cells within the tumor tissue, and higher expression of PD-1 on these T cells than the other groups in vivo. However, the SCC2 group showed no response to anti-PD-1 therapy. Transcriptome analysis revealed that the SCC2 group exhibited increased expression of genes related to GC (Hsd11b1, Sgk3, Tgfbr2, and Il7r) compared to SCC2-anti-PD-1 treated tumors. CONCLUSIONS: GC pathway activation is related to resistance to anti-PD-1 therapy.

CD69+ Vδ1γδ T cells are anti-tumor subpopulations in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC), one of the malignancies with a wide expression of stress ligands recognized by Vδ1γδ T cells, has received much attention in adoptive immunotherapy of γδ T cells. In this study, we aimed to identify the potential anti-tumor Vδ1γδ T subpopulations in HCC. METHODS: Healthy donors (HDs) and HCC patients were recruited from the Affiliated Cancer Hospital of Zhengzhou University. Blood and tumor tissue samples were obtained respectively. Bioinformatics methods were used to analyze total γδ T cells and subsets infiltration, overall survival of HCC patients with high and low infiltration level of Vδ1γδ T cells, and IFNG, granzyme A, granzyme B and perforin expression in TRDV1high/lowCD69high/low groups. CD69 expression and Vδ1γδT cells infiltration in HCC were detected by immunofluorescence. Phenotypic analysis of Vδ1γδ T cells in blood and tumor tissue samples were performed by flow cytometry. RESULTS: Vδ1γδ T cells infiltrating in HCC were associated with better clinical outcome. Study in tumor micro-environment (TME) of HCC demonstrated that not total Vδ1γδ T but CD69+ Vδ1γδ subset infiltration was associated with smaller tumor volume. Moreover, HCC patients simultaneously with high TRDV1 and CD69 expression produced more effector molecules and had longer survival time. Since Vδ1γδ T cells in the tumor microenvironment were often difficult to access, we demonstrated that CD69+ Vδ1γδ T cells also existed in peripheral blood mononuclear cells (PBMC) of HCC and displayed enhanced cytotoxic potentials than HDs. Finally, we investigated the functions and found that CD69+ Vδ1γδ T cells exhibited stronger tumor reactivities when challenged by tumor cells. CONCLUSIONS: CD69+ Vδ1γδ T cells are functional Vδ1γδ T cell subsets in patients with HCC. Circulating CD69+ Vδ1γδ T cell is a promising candidate in immunotherapy of HCC.

The inter-link of ageing, cancer and immunity: findings from real-world retrospective study.

BACKGROUND: Although the concept of declined immune function associated with cancer has been accepted extensively, real-world clinical studies focusing on analysis of the peripheral blood immune changes underlying ageing, immunity and cancer are scarce. METHODS: In this case-control study, we retrospectively analysed 1375 cancer patients and enrolled 275 age and gender matched healthy individuals. Flow cytometry was conducted to assess the immune changes. Further analysis was examined by SPSS 17.0 and GraphPad Prism 9 software. RESULTS: Cancer patients showed obviously decreased CD3+ T, CD3+CD4+ Th, CD3+CD8+ CTL, CD19+ B, CD16+CD56+ NK cell counts and lower percentage of PD-1 (programmed cell death protein-1, PD-1) positive cells than healthy control (P < 0.0001). For cancer patients, the reference range of circulating percentage of PD-1+CD45+ cells, PD-1+CD3+ T cells, PD-1+CD3+CD4+ Th cells and PD-1+CD3+CD8+ CTL (Cytotoxic T Lymphocyte, CTL) were 11.2% (95% CI 10.8%-11.6%), 15.5% (95% CI 14.7%-16.0%), 15.4% (95% CI 14.9%-16.0%) and 14.5% (95% CI 14.0%-15.5%), respectively. Moreover, the reduction of CD3+ T, CD3+CD4+ Th, CD3+CD8+ CTL, CD19+ B cell counts accompanied with age and stage advancing (P < 0.05). CD16+CD56+ NK cells decreased with stage, but elevated in aged and male cancer patients (P < 0.05). Additionally, the percentage of PD-1 positive cells varied across cancer types, raised with age and stage. Head and neck, pancreatic, gynaecological and lung demonstrated a higher level of the percentage of PD-1 positive cells than melanoma, prostate, and breast cancer (P < 0.05). CONCLUSIONS: This study provides the reference range of the percentage of PD-1 positive cells on peripheral blood, confirms the decreased immune cells and a series of immune changes accompanying with cancer, expands our real world evidence to better understand the interactions of ageing, cancer and immunity. Moreover, the circulating percentage of PD-1 positive cells shows similar tumor type distribution with tumor mutational burden (TMB), supports that it maybe a potential predictive biomarker for immune checkpoint inhibitor therapy.

In vivo growth of subclones derived from Lewis lung carcinoma is determined by the tumor microenvironment.

Heterogeneity is a fundamental feature of human tumors and plays a major role in drug resistance and disease progression. In the present study, we selected single-cell-derived cell lines (SCDCLs) derived from Lewis lung carcinoma (LLC1) cells to investigate tumorigenesis and heterogeneity. SCDCLs were generated using limiting dilution. Five SCDCLs were subcutaneously injected into wild-type C57BL/6N mice; however, they displayed significant differences in tumor growth. Subclone SCC1 grew the fastest in vivo, whereas it grew slower in vitro. The growth pattern of SCC2 was the opposite to that of SCC1. Genetic differences in these two subclones showed marked differences in cell adhesion and proliferation. Pathway enrichment results indicate that signal transduction and immune system responses were the most significantly altered functional categories in SCC2 cells compared to those in SCC1 cells in vitro. The number and activation of CD3+ and CD8+ T cells and NK cells in the tumor tissue of tumor-bearing mice inoculated with SCC2 were significantly higher, whereas those of myeloid cells were significantly lower, than those in the SCC1 and LLC1 groups. Our results suggest that the in vivo growth of two subclones derived from LLC1 was determined by the tumor microenvironment rather than their intrinsic proliferative cell characteristics.

TIGIT-expressing zoledronate-specific γδ T cells display enhanced antitumor activity.

Human γδ T cells hold a pivotal role in tumor immunosurveillance through their prompt activation and cytokine secretion and have received much attention in adoptive immunotherapy of clear cell renal cell carcinoma (ccRCC). However, the therapeutic effects are limited in ccRCC. Therefore, it is now critical to improve therapeutic strategies based on γδ T cells, especially identification of functional γδ T cell subsets. In this study, we aimed to identify γδ T cells that might have enhanced responses against ccRCC. Bioinformatic analysis showed that ccRCC patients with high T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) expression had higher levels of effector molecules. Then, we examined the changes in the TIGIT+ γδ T cell percentages of 6 ccRCC patients and 14 healthy subjects through zoledronate (ZOL) stimulation. Results indicated that percentages of TIGIT+ γδ T cells were positively correlated with activated γδ T cells in early activation stage. Further study demonstrated that TIGIT+ γδ T cells exhibited enhanced activation, contained more terminally differentiated effector γδ T cells and produced higher cytokine compared with TIGIT- γδ T cells. Finally, we investigated the functions and found that TIGIT+ γδ T cells exhibited stronger tumor reactivities and higher cytotoxicity when challenged by tumor cells. Above results imply that TIGIT+ γδ T cells are the main effectors in ZOL recognition and tumor cells challenging. The results of the present study serve as basis for future functional studies on TIGIT+ γδ T cells and provide a promising approach of immunotherapy in ccRCC.

Culturing adequate CAR-T cells from less peripheral blood to treat B-cell malignancies.

OBJECTIVE: Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure. METHODS: We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3+ T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells. RESULTS: The CAR-T cells were expanded to 1-3 × 108 cells in 8-10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma. CONCLUSIONS: Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50-100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.

Serum Sp17 Autoantibody Serves as a Potential Specific Biomarker in Patients with SAPHO Syndrome.

SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome shows a wide variability in musculoskeletal and cutaneous manifestations, and it is therefore underrecognized and misdiagnosed in the clinic due to a lack of specific markers. In this study, we aimed to identify specific biomarkers by screening serum autoantibodies in SAPHO patients with a 17K human whole-proteome microarray. The serum anti-Sp17 autoantibody was identified and verified to be a specific biomarker in patients with SAPHO syndrome. Indeed, the level of the anti-Sp17 autoantibody was significantly increased in patients with active SAPHO compared to patients with an inactive disease and healthy controls (P < 0.05). Additionally, serum anti-Sp17 autoantibody levels correlated with those of serum hypersensitive C-reactive protein (hsCRP), the erythrocyte sedimentation rate (ESR), and ß-crosslaps (ß-CTx) in patients with active SAPHO disease. Moreover, anti-Sp17 autoantibody levels were markedly decreased after anti-inflammatory treatment with pamidronate disodium, which downregulated levels of hsCRP and ESR in patients with active SAPHO. Thus, serum levels of the anti-Sp17 autoantibody might serve as a specific biomarker for the diagnosis of SAPHO syndrome or for monitoring the disease status.

Chaperonin-containing T-complex Protein 1 Subunit ζ Serves as an Autoantigen Recognized by Human Vδ2 γδ T Cells in Autoimmune Diseases.

Human γδ T cells recognize conserved endogenous and stress-induced antigens typically associated with autoimmune diseases. However, the role of γδ T cells in autoimmune diseases is not clear. Few autoimmune disease-related antigens recognized by T cell receptor (TCR) γδ have been defined. In this study, we compared Vδ2 TCR complementarity-determining region 3 (CDR3) between systemic lupus erythematosus (SLE) patients and healthy donors. Results show that CDR3 length distribution differed significantly and displayed oligoclonal characteristics in SLE patients when compared with healthy donors. We found no difference in the frequency of Jδ gene fragment usage between these two groups. According to the dominant CDR3δ sequences in SLE patients, synthesized SL2 peptides specifically bound to human renal proximal tubular epithelial cell line HK-2; SL2-Vm, a mutant V sequence of SL2, did not bind. We identified the putative protein ligand chaperonin-containing T-complex protein 1 subunit ζ (CCT6A) using SL2 as a probe in HK-2 cell protein extracts by affinity chromatography and liquid chromatography-electrospray ionization-tandem mass spectrometry analysis. We found CCT6A expression on the surface of HK-2 cells. Cytotoxicity of only Vδ2 γδ T cells to HK-2 cells was blocked by anti-CCT6A antibody. Finally, we note that CCT6A concentration was significantly increased in plasma of SLE and rheumatoid arthritis patients. These data suggest that CCT6A is a novel autoantigen recognized by Vδ2 γδ T cells, which deepens our understanding of mechanisms in autoimmune diseases.

TCRγ4δ1-engineered αßT cells exhibit effective antitumor activity.

T cell engineering with T cell receptors (TCRs) specific for tumors plays an important role in adoptive T-cell transfer (ATC) therapy for cancer. Here, we present a novel strategy to redirect peripheral blood-derived αßT cells against tumors via TCRγ4δ1 gene transduction. The broad-spectrum anti-tumor activity of TCRδ1 cells in innate immunity is dependent on CDR3δ1. TCRγ4δ1-engineered αßT cells were prepared by lentiviral transduction and characterized by analyzing in vitro and in vivo cytotoxicity to tumors, ability of proliferation and cytokine production, and their potential role in autoimmunity. Results show TCRγ4δ1 genes were transduced to approximately 36% of polyclonal αßT cells. TCRγ4δ1-engineered αßT cells exhibited an effective in-vitro TCRγδ-dependent cytotoxicity against various tumor cells via the perforin-granzyme pathway. They also showed a strong proliferative capacity and robust cytokine production. TCRγ4δ1-engineered αßT cells neither expressed mixed TCR dimers nor bound/killed normal cells in vitro. More importantly, adoptive transfer of TCRγ4δ1-engineered αßT cells into nude mice bearing a human HepG2 cell line significantly suppressed tumor growth. Our results demonstrate a novel role for TCRγ4δ1 in gene therapy and ATC for cancer.