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.

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.

An effective peptide vaccine strategy circumventing clonal MHC heterogeneity of murine myeloid leukaemia.

BACKGROUND: Therapeutic cancer vaccines are an attractive approach for treating malignant tumours, and successful tumour eradication depends primarily on controlling tumour immunosuppression status as well as heterogeneity of tumour cells driven by epigenetic alterations. METHODS: Peptide-loaded dendritic cell (DC) prime and non-infectious peptide booster heterologous immunisations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides. Heterologous vaccination regimen targeting multiple shared tumour antigens simultaneously with PD-L1 blockade was assessed against murine myeloid leukaemia. RESULTS: A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumours. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukaemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumour eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukaemia. CONCLUSIONS: Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimisation of cancer vaccines for the treatment of numerous viral diseases and malignant tumours.

Comparison and correlation among in vitro and in vivo assays to assess cord blood quality according to delivery temperature and time after collection.

Cord blood (CB) has been used as an alternative source for unrelated allogeneic hematopoietic stem cell transplantation. To determine which assay was useful for predicting the successful outcome of CB transplantation, CBs were grouped according to the temperature (4 °C, 24 °C, and 37 °C) and time (24, 48, and 72 h) after collection. The viability, early apoptosis, and colony forming units (CFUs) were ascertained for the total nucleated cells (TNCs) and CD34+ cells; in addition, the engraftment of infused CD34+ cells in NSG mice was determined. The viability of the TNCs and CD34+ cells and total CFUs were significantly decreased whereas the early apoptosis was significantly increased in the 72 h group at 37 °C compared to that of the 24 h group at 24 °C. The viability and early apoptosis of the TNCs correlated with those of CD34+ cells. In addition, the viability and early apoptosis correlated with the number of granulocyte/monocyte progenitor CFUs. In transplanted NSG mice, the frequency of human CD45+ cells decreased in the 72 h group at 24 °C compared to that of the 24 h group at 24 °C and was negatively correlated with early apoptosis of TNCs and CD34+ cells. This study demonstrated that the early apoptosis of TNCs and CD34+ cells constitutes a useful marker for predicting the engraftment of HSCs and may provide helpful data for standard assessment regarding CB quality by analyzing the correlation between in vitro and in vivo assays using NSG mice.

Ex Vivo Generated Human Cord Blood Myeloid-Derived Suppressor Cells Attenuate Murine Chronic Graft-versus-Host Diseases.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with anti-inflammatory activity, and expanded murine MDSCs are capable of attenuating preclinical acute graft-versus-host disease (aGVHD) severity. Two murine cGVHD models were used to evaluate the effectiveness of ex vivo cultured human cord blood (hCB) MDSCs in chronic GVHD (cGVHD). First, GVHD recipients surviving in a classic C57BL/6 into MHC-mismatched BALB/c aGVHD model developed cGVHD. Second, donor pretreatment with granulocyte colony-stimulating factor (G-CSF) induced cGVHD. hCB-MDSCs (1 × 106) were intravenously injected to determine their preventive effects (on days 5, 7, 10, and 21) or therapeutic effects (on days 21, 28, and 35). In the first model the onset of clinical cutaneous cGVHD was significantly delayed in preventive hCB-MDSCs-treated allogeneic recipients. Pathologic scoring of target organs confirmed these clinical results. Importantly, thymic tissues of GVHD mice treated with hCB-MDSCs were less severely damaged, showing higher numbers of double (CD4 and CD8) positive T cells with reduced expansion of donor-type CD4 and CD8 T cells. Moreover, late infusion of hCB-MDSCs controlled the severity of established cGVHD that had occurred in control recipients. In the second model, cGVHD induced by G-CSF-mobilized stem cell graft was associated with promotion of Th 17 and Th 2 differentiation. hCB-MDSCs attenuated clinical and pathologic cGVHD severity. Increased production of IL-17 and more infiltration of T cells and macrophages in cGVHD mice were markedly reduced after hCB-MDSCs treatment. Importantly, Foxp3+ regulatory T cells and IFN-γ-producing T cells were expanded, whereas IL-17- and IL-4-producing T cells were decreased in allogeneic recipients of hCB-MDSCs. Taken together, these results showed that hCB-MDSCs have preclinical capability of attenuating cGVHD by preserving thymus function and regulating Th 17 signaling, suggesting a possible therapeutic strategy for clinical application.

A novel Epstein-Barr virus-latent membrane protein-1-specific T-cell receptor for TCR gene therapy.

BACKGROUND: Adoptive transfer of genetically engineered T-cells to express antigen-specific T-cell receptor (TCR) is a feasible and effective therapeutic approach for numerous types of cancers, including Epstein-Barr virus (EBV)-associated malignancies. Here, we describe a TCR gene transfer regimen to rapidly and reliably generate T-cells specific to EBV-encoded latent membrane protein-1 (LMP1), which is a potential target for T-cell-based immunotherapy. METHODS: A novel TCR specific to LMP1 (LMP1-TCR) was isolated from HLA-A*0201 transgenic mice that were immunised with the minimal epitope LMP1166 (TLLVDLLWL), and LMP1-TCR-transduced peripheral blood lymphocytes were evaluated for functional specificities. RESULTS: Both human CD8 and CD4 T-cells expressing the LMP1-TCR provoked high levels of cytokine secretion and cytolytic activity towards peptide-pulsed and LMP1-expressing tumour cells. Notably, recognition of these T-cells to peptide-pulsed cells was maintained at low concentration of peptide, implying that the LMP1-TCR has high avidity. Infusion of these engineered T-cells revealed remarkable therapeutic effects and inhibition of tumour growth in a preclinical xenogeneic model. We observed explosive ex vivo proliferation of functional TCR-transduced T-cells with artificial antigen-presenting cells that express co-stimulatory molecules CD80 and 4-1BBL. CONCLUSIONS: These data suggest that the novel TCR-targeting LMP1 might allow the potential design of T-cell-based immunotherapeutic strategies against EBV-positive malignancies.

Long-term Outcome of Extranodal NK/T Cell Lymphoma Patients Treated With Postremission Therapy Using EBV LMP1 and LMP2a-specific CTLs.

Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.

A multimeric carcinoembryonic antigen signal inhibits the activation of human T cells by a SHP-independent mechanism: a potential mechanism for tumor-mediated suppression of T-cell immunity.

Carcinoembryonic antigen (CEA) is a well-known tumor antigen that is found in the serum of patients with various cancers and is correlated with an increased risk of cancer recurrence and metastasis. To understand the tumor environment and to develop antitumor therapies, CEA has been studied as an antigen to activate/tolerate specific T cells. In this study, we show that CEA can function as a coinhibitory molecule and can inhibit the activation of human peripheral blood mononucleated cell-derived T cells. The addition of CEA-overexpressing tumor cells or immobilized CEA dampened both cell proliferation and the expression of IL-2 and CD69 expression in T cells after TCR stimulation. The phosphorylation of ERK and translocation of NFAT were hampered in these cells, whereas the phosphorylation of proximal TCR signaling molecules such as ZAP70 and phospholipase Cγ was not affected by immobilized CEA. To determine the relevance of carcinoembryonic antigen-related cell adhesion molecule-1 and Src homology region 2 domain-containing phosphatase (SHP) molecules to CEA-mediated suppression, we tested the effect of the SHP inhibitor, NSC-87877, on CEA-mediated suppression of T cells; however, it did not reverse the effect of CEA. Collectively, these results indicate that CEA can function as a modulator of T-cell responses suggesting a novel mechanism of tumor evasion.

Development of cost-effective and fast KIR genotyping by multiplex PCR-SSP.

Killer-cell immunoglobulin-like receptors (KIR) control natural killer (NK) cell functions by recognizing HLA molecules and modulating the activity of NK cells. The KIR gene cluster contains polymorphic and highly homologous genes. Diversity of the KIR region is achieved through differences in gene content, allelic polymorphism, and gene copy number, which result in unrelated individuals having different KIR genotypes and individualized immune responses that are relevant to multiple aspects of human health and disease. Therefore, KIR genotyping is increasingly used in epidemiological studies. Here, we developed multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP) to compensate for the shortcomings of the conventional PCR-SSP method, which is most commonly used for KIR analysis. Multiplex PCR-SSP method involves six multiplex reactions that detect 16 KIR genes and distinguish variant types of some KIR genes by adding two reactions. The assay was evaluated in a blind survey using a panel of 40 reference DNA standards from the UCLA KIR Exchange Program. The results are 100% concordant with the genotype determined using Luminex-based reverse sequence-specific oligonucleotide typing systems. Additionally, we investigated the currently known 16 KIR genes and their common variants in 120 unrelated Korean individuals. The results were consistent with the KIR genotype previously reported by Hwang et al. This multiplex PCR-SSP is an efficient method for analyzing KIR genotypes in both small- and large-scale studies with minimal labor, reagents, and DNA. Furthermore, by providing a better definition of KIR polymorphisms it can contribute to developments in immunogenetics.

Association of KIR Genes with Middle East Respiratory Syndrome Coronavirus Infection in South Koreans.

BACKGROUND: Middle East respiratory syndrome (MERS) is a lower respiratory tract disease caused by a beta coronavirus (CoV) called MERS-CoV, characterized by a high mortality rate. We aimed to evaluate the association between genetic variation in killer cell immunoglobulin-like receptors (KIRs) and the risk of MERS in South Koreans. METHODS: KIR genes were genotyped by multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP). A case-control study was performed to identify the odds ratios (OR) of KIR genes for MERS and the association of KIR genes and their ligands, human leukocyte antigens (HLA) genes. RESULTS: KIR2DS4D and KIR3DP1F showed higher frequencies in the group of all patients infected with MERS-CoV than in the control group (p = 0.023, OR = 2.4; p = 0.039, OR = 2.7). KIR2DL1, KIR2DP1, and KIR3DP1D were significantly associated with moderate/mild (Mo/Mi) cases. KIR2DL2, KIR2DS1, and KIR3DP1F were affected in severe cases. When we investigated the association between KIR genes and their ligands in MERS patient and control groups, KIR3DL1+/Bw4(80I)+, KIR3DL1+/Bw6+, KIR3DL1+/Bw6-, KIR2DS1+/C2+, and KIR3DS+/Bw4(80I)+ were associated with MERS. KIR3DL1+/Bw6- was found in Mo/Mi cases. KIR2DS1+/C2+ and KIR2DS2+/C1+ were found in severe cases. CONCLUSION: Further investigations are needed to prove the various immune responses of MERS-CoV-infected cells according to variations in the KIR gene and ligand gene. A treatment strategy based on current research on the KIR gene and MERS-CoV will suggest potential treatment targets.

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.

Combinatorial molecular marker assays of WT1, survivin, and TERT at initial diagnosis of adult acute myeloid leukemia.

High levels of expression of Wilms' tumor gene 1 (WT1), survivin, or telomerase reverse transcriptase (TERT) genes are introduced as leukemia-associated targets predicting clinical outcome. We prospectively investigated the leukemia-associated gene transcripts by real-time quantitative polymerase chain reaction from 151 adult patients with AML associated with the patients' clinical characteristics. The maximum levels of each gene in bone marrow were 64.4-, 8.1-, and 3.9-fold higher than those in the normal control, respectively. In contrast to the WT1 and TERT levels, survivin showed comparatively higher expression in the unfavorable cytogenetic group of patients. We found a significant difference in survivin levels between the CR and non-CR groups (P = 0.0237). TERT expression levels were higher in patients who had a greater number of peripheral blood leukemic blasts at diagnosis (P = 0.0191). Non-MRC subtypes and patients without specific mutations were the most powerful predictive factors for a better CR rate, by multivariate analyses. The lower levels of both WT1 and survivin co-expression (P = 0.0129) and both survivin + TERT co-expression (P = 0.0115) were significant factors for better OS. Besides lower initial levels of serum ferritin (P = 0.0401), lower levels of WT1 (P = 0.0438) and survivin (P = 0.0401), lower levels of both WT1 and survivin co-expression (P = 0.0031), and the three-gene combination of lower WT1 + survivin + TERT (P = 0.0454) were powerful predictive factors for better EFS. As our findings were based on a single disease entity, that is, adult AML, they suggest that the expression of these genes may be critical for the immunobiology of AML to influence the clinical outcome in various ways.

Response to pretransplant hypomethylating agents influences the outcome of allogeneic hematopoietic stem cell transplantation in adults with myelodysplastic syndromes.

This study describes a retrospective analysis on the transplant outcome of 56 consecutive patients with myelodysplastic syndrome (MDS) according to their response to hypomethylating agents (HMA). While 2-yr disease-free survival (DFS) of patients who transformed to acute myeloid leukemia (n = 12) was 25%, that of the remaining patients with MDS according to response to HMA was 73.1%, 68.1%, 50.0%, and 20.8% in G-COR (group of continuous response, n = 19), G-NoC (group of no change, n = 15), G-LOR (group of loss of response, n = 6), and G-DP (group of disease progression, n = 4), respectively. When dichotomized as G-COR/G-NoC versus G-LOR/G-DP, significantly different 2-yr DFS (71.0% vs. 33.3%; P = 0.004) and relapse (14.1% vs. 46.7%; P = 0.016) were demonstrated. On multivariate analysis, G-LOR/G-DP [hazard ratio (HR), 3.91; P = 0.008] and poor karyotype at transplantation (HR, 2.69; P = 0.017) were the significant predictors for poor DFS, as G-LOR/G-DP was for relapse (HR, 6.28; P = 0.011). DFS was significantly poor in patients with any of the two predictors in all MDS (81.5% vs. 34.9%; P = 0.001) or higher-risk MDS (HrMDS) at the time of HMA (80.7% vs. 29.2%; P = 0.005). G-COR showed a trend of better DFS compared with G-NoC among HrMDS (74.6% vs. 36.5%; P = 0.090). These results implicate the significance of response to HMA on hematopoietic stem cell transplantation (HSCT) outcomes and support the need for future study to verify the suggested strategy of proceeding to transplantation before LOR or DP, especially for HrMDS.

Shared epitope and radiologic progression are less prominent in elderly onset RA than young onset RA.

The aim of this study was to determine the influence of HLA-DRB1 and HLA-DQB1 genes on the disease susceptibility and the disease severity in elderly onset rheumatoid arthritis (EORA) compared with young onset rheumatoid arthritis (YORA) in Korean patients. Genetic analysis of HLA-DRB1 and HLA-DQB1 alleles was performed in three groups. Group 1 included 63 patients who were diagnosed with (rheumatoid arthritis) RA after the age of 60 (EORA). Group 2 consisted of 109 patients who were diagnosed with RA before the age of 60 (YORA). Group 3 involved 133 normal controls. The shared-epitope-coding alleles included the members of the HLA-DRB1*04 allele group (*0401, *0404, *0405, *0408, *0410), HLA-DRB1*01 allele group (*0101,*0102), HLA-DRB1*1001, and HLA-DRB1*1402. The disease severity was assessed by the modified total sharp score (mTSS). The shared-epitope-coding alleles were more frequently observed in the RA patients than in the normal controls. The shared-epitope-coding alleles were less frequently found in EORA group than YORA group (31/63 (49.2%) in group 1, 72/109 (66.1%) in group 2, 45/133 (33.8%) group 3, p = 0.02). Although the mTSS of the group 1 was higher than group 2 at symptom onset, the overall mean mTSS of the group 1 was lower than that of group 2 (26.8 vs. 57.5, p < 0.05). HLA-DQ*04 showed the higher frequency in the patients group than in normal controls (p < 0.001). And HLA-DQ*04 was less commonly found in the patients with EORA than YORA (p < 0.05). The influence of shared epitope and HLA-DQ*04 alleles may be less significant on disease susceptibility in EORA. The presence of shared-epitope-coding alleles did not appear to influence on disease severity in EORA patients as well as in YORA patients. Radiologic deterioration in EORA group was less severe than in YORA group. The presence of shared epitope and radiologic progression are less prominent in EORA patients than YORA patients.

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.

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.

Comprehensive analysis of chemokine gene polymorphisms in Korean children with autoimmune thyroid disease.

Chemokines are chemotactic cytokines that can cause directed migration of leukocytes. The aim of this study was to examine differences in single nucleotide polymorphisms (SNP) of chemokine in AITD patients compared to normal controls. A total of 86 Korean pediatric patients were included in the patient group and 183 adults were included in the normal control group. To compare influences of several chemokine gene polymorphisms, 25 SNPs in 16 chemokine genes were analyzed. Genotype frequencies of CCL11(rs3744508)AA(OR = 6.9) and CCR2(rs1799864)AA(OR = 3.8) were higher in the AITD patients than in the controls, whereas CCL17(rs223828)CC was lower in the AITD patients than in the controls(OR = 0.4). In comparison between Graves' disease (GD) patients and controls, genotype frequency of CCL17(rs223828)CC(OR = 0.4) was lower in the GD group, whereas those of CCR2(rs1799864)AA(OR = 4.8) were higher in the GD group. The genotype frequency of CCL11(rs3744508)AA(OR = 11.3) was higher in Hashimoto's thyroiditis (HT) patients, whereas that of CXCL8(rs2227306)CC(OR = 0.4) was lower in HT patients. Polymorphisms of CCL11(rs3744508), CCL17(rs223828), and CCR2(rs1799864) might be associated with AITD, with CCL17(rs223828), CCR2(rs1799864) and CXCR2(rs2230054, rs1126579) affecting GD and CCL11(rs3744508) and CXCL8(rs2227306) affecting HT in Korean children.

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.

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.