Prediction of TKI response in EGFR-mutant lung cancer patients-derived organoids using malignant pleural effusion.

Patient-derived organoids (PDOs) are valuable in predicting response to cancer therapy. PDOs are ideal models for precision oncologists. However, their practical application in guiding timely clinical decisions remains challenging. This study focused on patients with advanced EGFR-mutated non-small cell lung cancer and employed a cancer organoid-based diagnosis reactivity prediction (CODRP)-based precision oncology platform to assess the efficacy of EGFR inhibitor treatments. CODRP was employed to evaluate EGFR-tyrosine kinase inhibitors (TKI) drug sensitivity. The results were compared to those obtained using area under the curve index. This study validated this index by testing lung cancer-derived organoids in 14 patients with lung cancer. The CODRP index-based drug sensitivity test reliably classified patient responses to EGFR-TKI treatment within a clinically suitable 10-day timeline, which aligned with clinical drug treatment responses. This approach is promising for predicting and analyzing the efficacy of anticancer, ultimately contributing to the development of a precision medicine platform.

Human gamma-delta (γδ) T cell therapy for glioblastoma: A novel alternative to overcome challenges of adoptive immune cell therapy.

Glioblastoma is the most common brain malignancy with devastating prognosis. Numerous clinical trials using various target therapeutic agents have failed and recent clinical trials using check point inhibitors also failed to provide survival benefits for glioblastoma patients. Adoptive T cell transfer is suggested as a novel therapeutic approach that has exhibited promise in preliminary clinical studies. However, the clinical outcomes are inconsistent, and there are several limitations of current adoptive T cell transfer strategies for glioblastoma treatment. As an alternative cell therapy, gamma-delta (γδ) T cells have been recently introduced for several cancers including glioblastoma. Since the leading role of γδ T cells is immune surveillance by recognizing a broad range of ligands including stress molecules, phosphoantigens, or lipid antigens, recent studies have suggested the potential benefits of γδ T cell transfer against glioblastomas. However, γδ T cells, as a small subset (1-5%) of T cells in human peripheral blood, are relatively unknown compared to conventional alpha-beta (αß) T cells. In this context, our study introduced γδ T cells as an alternative and novel option to overcome several challenges regarding immune cell therapy in glioblastoma treatment. We described the unique characteristics and advantages of γδ T cells compared to conventional αß T cells and summarize several recent preclinical studies using human gamma-delta T cell therapy for glioblastomas. Finally, we suggested future direction of human γδ T cell therapy for glioblastomas.

Polymorphisms of Killer Ig-like Receptors and the Risk of Glioblastoma.

PURPOSE: The immune responses of natural killer (NK) cells against cancer cells vary by patient. Killer Ig-like receptors (KIRs), which are some of the major receptors involved in regulating NK cell activity for killing cancer cells, have significant genetic variation. Numerous studies have suggested a potential association between the genetic variation of KIR genes and the risk of development or prognosis of various cancer types. However, an association between genetic variations of KIR genes and glioblastoma (GB) remains uncertain. We sought to evaluate the association of genetic variations of KIRs and their ligand genes with the risk of GB development in Koreans. METHODS: A case-control study was performed to identify the odds ratios (ORs) of KIR genes and Classes A, B, and, C of the human leukocyte antigen (HLA) for GB. The GB group was comprised of 77 patients with newly diagnosed IDH-wildtype GB at our institution, and the control group consisted of 200 healthy Korean volunteers. RESULTS: There was no significant difference in the frequency of KIR genes and KIR haplotypes between the GB and control groups. Genetic variations of KIR-2DL1, 3DL1, and 3DS1 with their ligand genes (HLA-C2, HLA-Bw4/6, and Bw4, respectively) had effects on the risk of GB in Korean patients. The frequency of KIR-2DL1 with HLA-C2 (OR 2.05, CI 1.19-3.52, p = 0.009), the frequency of KIR-3DL1 without HLA-Bw4 (80I) (OR 8.36, CI 4.06-17.18, p < 0.001), and the frequency of KIR-3DL1 with Bw6 (OR 4.54, CI 2.55-8.09, p < 0.001) in the GB group were higher than in the control group. In addition, the frequency of KIR-2DL1 without HLA-C2 (OR 0.44, CI 0.26-0.75, p = 0.003), the frequency of KIR-3DL1 with HLA-Bw4 (80T) (OR 0.13, CI 0.06-0.27, p < 0.001), the frequency of KIR-3DL1 without Bw6 (OR 0.27, CI 0.15-0.49, p < 0.001), and the frequency of KIR-3DS1 with Bw4 (80I) (OR 0.03, CI 0.00-0.50, p < 0.001) in the GB group were lower than in the control group. CONCLUSIONS: This study suggests that genetic variations of KIRs and their ligand genes may affect GB development in the Korean population. Further investigations are needed to demonstrate the different immune responses for GB cells according to genetic variations of KIR genes and their ligand genes.

CD40 ligand stimulation affects the number and memory phenotypes of human peripheral CD8+ T cells.

BACKGROUND: CD40L is primarily expressed on activated CD4+ T cells and binds to CD40 which is expressed by various cells including dendritic cells, macrophages and B lymphocytes. While CD40-CD40L interaction is known to be direct between B cells and CD4+ T cells which results in proliferation and immunoglobulin isotype switching, antigen presenting cells (APCs) were thought to be involved in the delivery of CD4+ help to CD8+ T cells by cross-talk between CD4+ and CD8+ T cells and APCs. However, subsequent study demonstrated that CD40L signal can be directly delivered to CD8+ T cells by CD40 expression on CD8+ T cells. Since most studies have been carried out in murine models, we aimed to investigate the direct effect of CD40L on human peripheral CD8+ T cells. RESULTS: Human peripheral CD8+ T cells were isolated to exclude the indirect effect of B cells or dendritic cells. Upon activation, CD40 expression on CD8+ T cells was transiently induced and stimulation with artificial APCs expressing CD40L (aAPC-CD40L) increased the number of total and central memory CD8+ T cells and also pp65 specific CD8+ T cells. Stimulation with aAPC-CD40L also resulted in higher proportion of central memory CD8+ T cells. CONCLUSIONS: Our study suggests that CD40L has an effect on the increased number of CD8+ T cells through CD40 expressed on activated CD8+ T cells and has influence on memory CD8+ T cell generation. Our results may provide a new perspective of the effect of CD40L on human peripheral CD8+ T cells, which differ according to the memory differentiation status of CD8+ T cells.

Comprehensive Analysis of Epstein-Barr Virus LMP2A-Specific CD8+ and CD4+ T Cell Responses Restricted to Each HLA Class I and II Allotype Within an Individual.

Latent membrane protein 2A (LMP2A), a latent Ag commonly expressed in Epstein-Barr virus (EBV)-infected host cells, is a target for adoptive T cell therapy in EBV-associated malignancies. To define whether individual human leukocyte antigen (HLA) allotypes are used preferentially in EBV-specific T lymphocyte responses, LMP2A-specific CD8+ and CD4+ T cell responses in 50 healthy donors were analyzed by ELISPOT assay using artificial Ag-presenting cells expressing a single allotype. CD8+ T cell responses were significantly higher than CD4+ T cell responses. CD8+ T cell responses were ranked from highest to lowest in the order HLA-A, HLA-B, and HLA-C loci, and CD4+ T cell responses were ranked in the order HLA-DR, HLA-DP, and HLA-DQ loci. Among the 32 HLA class I and 56 HLA class II allotypes, 6 HLA-A, 7 HLA-B, 5 HLA-C, 10 HLA-DR, 2 HLA-DQ, and 2 HLA-DP allotypes showed T cell responses higher than 50 spot-forming cells (SFCs)/5×105 CD8+ or CD4+ T cells. Twenty-nine donors (58%) showed a high T cell response to at least one allotype of HLA class I or class II, and 4 donors (8%) had a high response to both HLA class I and class II allotypes. Interestingly, we observed an inverse correlation between the proportion of LMP2A-specific T cell responses and the frequency of HLA class I and II allotypes. These data demonstrate the allele dominance of LMP2A-specific T cell responses among HLA allotypes and their intra-individual dominance in response to only a few allotypes in an individual, which may provide useful information for genetic, pathogenic, and immunotherapeutic approaches to EBV-associated diseases.

In Vitro Expansion of Vδ1+ T Cells from Cord Blood by Using Artificial Antigen-Presenting Cells and Anti-CD3 Antibody.

γδ T cells have the potential for adoptive immunotherapy since they respond to bacteria, viruses, and tumors. However, these cells represent a small fraction of the peripheral T-cell pool and require activation and proliferation for clinical benefits. In cord blood, there are some γδ T cells, which exhibit a naïve phenotype, and mostly include Vδ1+ T cells. In this study, we investigated the effect of CD3 signaling on cord blood γδ T-cell proliferation using K562-based artificial antigen presenting cells expressing costimulatory molecules. There were significantly more Vδ1+ T cells in the group stimulated with anti-CD3 antibody than in the group without. In cultured Vδ1+ T cells, DNAM-1 and NKG2D were highly expressed, but NKp30 and NKp44 showed low expression. Among various target cells, Vδ1+ T cells showed the highest cytotoxicity against U937 cells, but Daudi and Raji cells were not susceptible to Vδ1+ T cells. The major cytokines secreted by Vδ1+ T cells responding to U937 cells were Granzyme B, IFN-γ, and sFasL. Cytotoxicity by Vδ1+ T cells correlated with the expression level of PVR and Nectin of DNAM-1 ligands on the surface of target cells. Compared to Vδ2+ T cells in peripheral blood, cord blood Vδ1+ T cells showed varying cytotoxicity patterns depending on the target cells. Here, we determined the ideal conditions for culturing cord blood Vδ1+ T cells by observing that Vδ1+ T cells were more sensitive to CD3 signals than other subtypes of γδ T cells in cord blood. Cultured cord blood Vδ1+ T cells recognized target cells through activating receptors and secreted numerous cytotoxic cytokines. These results are useful for the development of tumor immunotherapy based on γδ T cells.

Distributions of 11-loci HLA alleles typed by amplicon-based next-generation sequencing in South Koreans.

The range of HLA typing for successful hematopoietic stem cell transplantation (HSCT) is gradually expanding with the next-generation sequencing (NGS)-based improvement in its quality. However, it is influenced by the allocation of finances and laboratory conditions. HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 alleles were genotyped at the 3-field level by amplicon-based NGS using MiSeqDx system and compared to our previous study employing long-range PCR and NGS using TruSight HLA v2 kit, in healthy donors from South Korea. Exon 2, exons 2/3, exons 2/3/4 or 5 of 11-loci were amplified by multiplex PCR. The sequence reads of over 53 depth counts were consistently obtained in each sample exon, depending on the target exon determined to match the reference sequence contained in the IPD-IMGT/HLA Database. HLA alleles were investigated by combinations of the determined exons. A total of 18 alleles with a frequency over 10% were found at the 11 HLA loci. Three ambiguities of HLA-A, -C, and -DRB1 were resolved. We observed a total of 26 HLA-A ~ C ~ B and 6 HLA-DRB1 ~ DQA1 ~ DQB1 ~ DPA1 ~ DPB1 haplotypes having significant linkage disequilibrium between alleles at all neighboring HLA loci. This result was compatible with the previous one, using TruSight HLA v2 kit. Advantages are simple and short progress time because one plate is used for each PCR step in one PCR machine and 11-loci HLA typing is possible even if only eight samples. These data suggested that expanded 11-loci HLA typing data by amplicon-based NGS might help perform HSCT.

Human allogenic γδ T cells kill patient-derived glioblastoma cells expressing high levels of DNAM-1 ligands.

Adoptive transfer of γδ T cells is a novel immunotherapeutic approach to glioblastoma. Few recent studies have shown the efficacy of γδ T cells against glioblastoma, but no previous studies have identified the ligand-receptor interactions between γδ T cells and glioblastoma cells. Here, we identify those ligand-receptor interactions and provide a basis for using γδ T cells to treat glioblastoma. Vγ9Vδ2 T cells were generated from peripheral blood mononuclear cells of healthy donors using artificial antigen presenting cells. MICA, ULBP, PVR and Nectin-2 expression in 10 patient-derived glioblastoma (PDG) cells were analyzed. The in vitro cytokine secretion from the γδ T cells and their cytotoxicity toward the PDG cells were also analyzed. The in vivo anti-tumor effects were evaluated using a U87 orthotopic xenograft glioblastoma model. Expression of ligands and cytotoxicity of the γδ T cells varied among the PDG cells. IFN-γ and Granzyme B secretion levels were significantly higher when γδ Tcells were co-cultured with high-susceptible PDG cells than when they were co-cultured with low-susceptible PDG cells. Cytotoxicity correlated significantly with the expression levels of DNAM-1 ligands of the PDG cells. Blocking DNAM-1 resulted in a decrease in γδ T cell-mediated cytotoxicity and cytokine secretion. Intratumoral injection of γδ T cells showed anti-tumor effects in an orthotopic mouse model. Allogenic γδ T cells showed potent anti-tumor effects on glioblastoma in a DNAM-1 axis dependent manner. Our findings will facilitate the development of clinical strategies using γδ T cells for glioblastoma treatment.

The HLA-B*07:457 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-B*07:457 differs from HLA-B*07:02:01:01 in codon 117 in exon 3.

The HLA-DRB1*12:97 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-DRB1*12:97 differs from HLA-DRB1*12:02:01:01 in codon 63 in exon 2.

Identification of Naturally Processed Epitope Region Using Artificial APC Expressing a Single HLA Class I Allotype and mRNA of HCMV pp65 Antigen Fragments.

Recently, long synthetic peptides or in silico-predicted epitope peptides have been used to identify T cell epitopes, but these approaches may not be suitable for investigating naturally processed epitopes. Here, mRNAs, including fragments or predicted epitope sequences of HCMV pp65 antigen, were generated by in vitro transcription following transcriptionally active PCR. Then, artificial antigen-presenting cells (aAPCs) expressing a single HLA allotype were transfected with mRNAs to identify epitopes in donors with T cell responses that recognize pp65 antigen restricted to HLA-A*02:01, -A*02:06, or -B*07:02. T cells restricted to a particular HLA allotype showed positive responses in some of the 10 fragment antigens. Among predicted epitopes within these positive fragments, three epitopes of HLA-A*02:01, -A*02:06, and -B*07:02 were confirmed. In addition, T cells expanded by anti-CD3 stimulation for two weeks could also be effectively used for the identification of these T cell epitopes, although there were individual differences. These results demonstrated that fragment antigens and epitopes can be rapidly generated using mRNA, and naturally processed antigenic regions can be detected using aAPCs without a T cell cloning procedure. This method will help to identify novel T cell epitopes for developing immunotherapy and vaccines against infectious diseases and cancer.

The HLA-B*51:353 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-B*51:353 differs from HLA-B*51:01:01:01 in codon 95 in exon 3.

HLA polymorphisms and risk of glioblastoma in Koreans.

PURPOSE: Immune responses for cancer cells can be altered according to genetic variation of human leukocyte antigen (HLA). Association of HLA polymorphism with risk of various cancer types is well known. However, the association between HLA and glioblastoma (GBM) remains uncertain. We sought to evaluate the association of HLA polymorphism with risk of GBM development in Koreans. MATERIALS AND METHODS: A case-control study was performed to identify the odds ratios (OR) of HLA class I and II genes for GBM. The control group consisted of 142 healthy Korean volunteers, and the GBM group was 80 patients with newly diagnosed GBM at our institution. HLA class I (-A, -B, and-C) and class II (-DR, -DQ, and-DP) genotyping was performed by high-resolution polymerase chain reaction (PCR)-sequence-based typing (PCR-SBT) methods. RESULTS: There were significantly decreased frequencies of HLA-A*26:02 (OR 0.22 CI 0.05-0.98), HLA-C*08:01 (OR 0.29 CI 0.10-0.87), and HLA-DRB1*08:03 (OR 0.32 CI 0.11-0.98), while there was significantly increased frequency of HLA-C*04:01 (OR 2.29 CI 1.05-4.97). In analysis of haplotypes, the frequency of DRB1*14:05-DQB1*05:03 was significantly decreased (OR 0.22 CI 0.05-0.98). CONCLUSION: This study suggests that genetic variations of HLA may affect GBM development in Koreans. Further investigations with larger sample sizes are needed to delineate any potential role of the HLA polymorphisms in the pathogenesis of GBM development.

γδ T cells cultured with artificial antigen-presenting cells and IL-2 show long-term proliferation and enhanced effector functions compared with γδ T cells cultured with only IL-2 after stimulation with zoledronic acid.

BACKGROUND AIMS: Immunotherapeutic approaches using γδ T cells have emerged as the function of γδ T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of γ9δ2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of γδ T cells have also been studied recently. However, there are no studies that directly compare γδ T cells cultured with phosphoantigens with those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of γδ T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-γδ T cells) and γδ T cells stimulated with only zoledronic acid (ZA) (ZA-γδ T cells). METHODS: Peripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-γδ T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-γδ T cells were stimulated with only IL-2. Cultured γδ T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-γδ T cells and ZA-γδ T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model. RESULTS: Fold expansion on day 21 was 690.7 ± 413.1 for ZA-γδ T cells and 1415.2 ± 1016.8 for aAPC- γδ T cells. Moreover, aAPC-γδ T cells showed continuous growth, whereas ZA-γδ T cells showed a decline in growth after day 21. The T-cell receptor Vγ9+δ2+ percentages (mean ± standard deviation) on day 21 were 90.0 ± 2.7% and 87.0 ± 4.5% for ZA-γδ T cells and aAPC-γδ T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-γδ T cells. In DEG analysis, aAPC-γδ T cells and ZA-γδ T cells formed distinct clusters, and aAPC-γδ T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-γδ T cells compared with ZA-γδ T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-γδ T-cell treatment increased survival, and a higher frequency of aAPC-γδ T cells was shown in bone marrow compared with ZA-γδ T cells. CONCLUSIONS: Overall, this study demonstrates that aAPC-γδ T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-γδ T cells and provides a basis for using aAPC-γδ T cells in further studies, including clinical applications and genetic engineering of γδ T cells.

Exosomes from human cord blood plasma accelerate cutaneous wound healing by promoting fibroblast function, angiogenesis, and M2 macrophage differentiation.

Exosomes contain natural cargo molecules, such as miRNA, mRNA, and proteins, and transfer these functional cargos to neighboring or distant cells through circulation. In the wound-healing process, exosomes in the human blood and body fluids perform various functions, including proliferation, angiogenesis, differentiation, and wound healing, owing to their unique compositions. However, there is very limited information on the wound-healing effect of proteins in human cord blood plasma exosomes (CBPexo). Therefore, we studied the wound-healing potential of these proteins in terms of fibroblast functions, angiogenesis, and M2 macrophage differentiation. When scratch wound assays were conducted using human fibroblasts, CBPexo exhibited better wound-healing effects than adult blood plasma exosomes (ABPexo). CBPexo also promoted angiogenesis and differentiation of M2 macrophages, thus promoting the transition from inflammation to proliferation. To evaluate the CBPexo molecules involved, five proteins, GAL-3, GAL-7, HSP-72, PIP, and S100-A7, were selected through proteomic analysis, and their functions were investigated using an artificial exosome that expresses these proteins. Among these, HSP72 and PIP exhibited wound-healing effects similar to CBPexo. Furthermore, artificial exosomes expressing both HSP72 and PIP showed better wound-healing effects than CBPexo. Therefore, the use of artificial CBPexo can potentially overcome the limitations related to exosome production from CB.

The HLA-B*13:144 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-B*13:144 differs from HLA-B*13:01:01:01 in codon 12 in exon 2.

The HLA-A*24:514N allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-A*24:514N differs from HLA-A*24:02:01:01 in codon 167 in exon 3.

The HLA-A*02:954 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-A*02:954 differs from HLA-A*02:06:01:01 in codon 82 in exon 2.

The HLA-DRB1*09:45 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-DRB1*09:45 differs from HLA-DRB1*09:01:02:01 in codon 19 in exon 2.

The HLA-A*11:384 allele identified in a volunteer donor for hematopoietic stem cell transplant.

HLA-A*11:384 differs from HLA-A*11:01:01:01 in codon 151 in exon 3.