Local Myeloid-Derived Suppressor Cells Impair Progression of Experimental Autoimmune Uveitis by Alleviating Oxidative Stress and Inflammation.

Purpose: To evaluate the immune regulatory effect of human cord blood myeloid-derived suppressor cells (MDSCs) in experimental autoimmune uveitis (EAU) models. Methods: MDSCs (1 × 106) or PBS were injected into established C57BL/6 EAU mice via the subconjunctival route on days 0 and 7. The severity of intraocular inflammation was evaluated for up to 3 weeks. Tissue injury and inflammation were analyzed using immunolabelled staining, real-time PCR, and ELISA. In addition, immune cells in draining lymph nodes (LNs) were quantified using flow cytometry. Results: After 21 days, the clinical scores and histopathological grades of EAU were lower in the MDSCs group compared with the PBS group. Local administration of MDSCs suppressed the oxidative stress and the expression of TNF-α and IL-1ß in the retinal tissues. In addition, it inhibited the activation of pathogenic T helper 1 (Th1) and Th17 cells in draining LNs. MDSCs increased the frequency of CD25+ Foxp3+ regulatory T cells and the mRNA expression of IL-10, as an immune modulator. Conclusions: MDSCs suppressed inflammation and oxidative stress in the retina and inhibited pathogenic T cells in the LNs in EAU. Therefore, ocular administration of MDSCs has therapeutic potential for uveitis.

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.

Local and Systemic Injections of Human Cord Blood Myeloid-Derived Suppressor Cells to Prevent Graft Rejection in Corneal Transplantation.

Myeloid-derived suppressor cells (MDSCs) are therapeutic agents to prevent graft rejection in organ transplants by modulating inflammation. Herein, the immunosuppressive effect of human cord blood MDSCs on corneal allograft models was confirmed. CB-MDSCs were locally (subconjuctival, 5 × 105) or systemically (intravenous, 1 × 106) injected twice on days 0 and 7. A corneal transplantation model was established using C57BL/6 and BALB/c mice, and corneal graft opacity was measured to evaluate graft rejection up to 6 weeks. Results showed that graft survival in the MDSCs groups increased compared to vehicle groups after 42 days. Systemic and local MDSC administration inhibited the maturation (MHC-IIhi CD11c+) of dendritic cells (DCs) and the differentiation of interferon γ+ CD4+ Th1 in draining lymph nodes (LNs). However, vehicle groups increased the infiltration of CD3+ T cells and F4/80+ macrophages and produced prominent neovascular and lymphatic vessels into the graft site with increased mRNA expression of VEGF-A/C and VEGFR-1/R-3. Local MDSCs administration showed prominent anti-angiogenic/anti-lymphangiogenic effects even at lower MDSCs doses. Thus, CB-MDSCs could relatively suppress the infiltration of pathological T cells/macrophages into the corneas and the migration of mature DCs into draining LNs Therefore, ocular and systemic MDSCs administration showed therapeutic potential for preventing corneal allograft rejection.

Comprehensive analysis of mycobacterium tuberculosis antigen-specific CD4+ T cell responses restricted by single HLA class II allotype in an individual.

Mycobacterium tuberculosis infection is generally asymptomatic as latent tuberculosis, but it is still known as the world's leading bacterial cause of death. The diagnosis of latent tuberculosis infection relies on the evidence of cellular immunity to mycobacterial antigens. Since the association between HLA class II and tuberculosis infection has been reported in several population groups, a detailed study on the CD4+ T cell response to major tuberculosis antigens is needed. To elucidate which HLA class II allotypes in an individual are preferentially used in tuberculosis, CD4+ T cells specific to TB10.4, Ag85b, ESAT-6, and CFP-10 of Mycobacterium tuberculosis antigens were analyzed comprehensively. A total of 33 healthy donors were analyzed by ex vivo and cultured ELISPOT using panels of artificial antigen-presenting cells expressing a single HLA class II allotype. The CD4+ T cell responses were increased by an average of 39-fold in cultured ELISPOT compared with ex vivo ELISPOT. In ex vivo and cultured ELISPOT, CD4+ T cell responses showed significantly higher by HLA-DR than those of HLA-DQ and HLA-DP locus. In cultured ELISPOT, 9 HLA-DR allotypes, 4 HLA-DQ allotypes, and 3 HLA-DP allotypes showed positive CD4+ T cell responses. Among ten donors with positive CD4+ T cell responses when tested for mixed Mycobacterium tuberculosis antigens, seven donors were positive for only a single allotype, and three were positive for two allotypes in an individual. However, only one allotype was used for a single antigen-specific response when a single tuberculosis antigen was used individually. These results on the distribution of HLA class II allotypes showing high CD4+ T-cell responses to Mycobacterium tuberculosis antigens and the intra-individual allotype dominance will provide valuable information for understanding the immunobiology and immunogenetics of tuberculosis, which can contribute to the development of more effective vaccines.

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.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II.

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016-2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.

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.

Extracellular vesicles from human umbilical cord blood plasma modulate interleukin-2 signaling of T cells to ameliorate experimental autoimmune encephalomyelitis.

Human umbilical cord blood (UCB) cell-derived extracellular vesicles (EV) reportedly play immunosuppressive roles; however, UCB plasma-derived extracellular vesicles (CBP EVs) remain poorly studied. We examined the immunosuppressive potential of CBP EVs compared to that of adult blood plasma-derived extracellular vesicles (ABP EVs) in vitro and constructed an experimental autoimmune encephalomyelitis (EAE) model. Methods: CBP EVs were isolated by ultracentrifugation and their proteomic profiling was performed using the high-resolution liquid chromatography with tandem mass spectrometry. Human T lymphocytes or mouse splenocytes labeled with carboxyfluorescein succinimidyl ester were incubated with CBP EV to measure the immunosuppressive function of CBP EV. The effect on T-cell polarization was analyzed by flow cytometry and enzyme-linked immunospot assay. The matrix metalloproteinase (MMP) function in CBP EV was specifically inhibited using a chemical inhibitor. The efficacy of CBP EVs in the EAE mouse model was determined by scoring the symptoms and analyzing cell phenotype and cytokines using mouse splenocytes. We generated genetically engineered artificial EVs using HLA/MIC-null HEK293T (H1ME-5) cell line to characterize the immunosuppressive effect of CBP EV. Results: CBP EVs primarily inhibited the proliferation of T cells by reducing the production of IL-2. Specifically, CBP EV-derived matrix metallopeptidase cleaved the IL-2 receptor α (CD25) on the surface of activated T cells, consequently downregulating IL-2 signaling in response to IL-2R engagement. Although the inhibition of MMP activity in CBP EVs abrogated CD25 cleavage and restored IL-2 production in activated T cells, the immunosuppressive response was not fully recovered. Thus, we further analyzed changes in immunosuppressive cells such as regulatory T cells and bone marrow-derived suppressor cells by CBP EV. Further, GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP were highly enriched in CBP EV-mimics in which they served as pivotal mediators of CBP EV-induced immunosuppressive effects. Therefore, we generated genetically engineered GAL-3, GAL-7, S100-A7, MMP-9, MMP-8, HSP-72, and PIP-EVs using HLA/MIC-null HEK293T cells to characterize the immunosuppressive effect of these molecules. Among these, MMP-9 and HSP-72-enriched EVs showed the most significant T cell immunosuppression. Conclusion: CBP EVs inhibited T cell proliferation and EAE development by modulating IL-2 signaling and immunosuppressive cell fate. CBP EVs contain critical components for immunosuppression and that CBP EV mimics, specifically those expressing MMP-9 and HSP-72, may offer a novel promising strategy for the treatment of various autoimmune diseases.

T Cells Modified with CD70 as an Alternative Cellular Vaccine for Antitumor Immunity.

PURPOSE: Successful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T cells. Dendritic cells (DCs) are well-known potent antigen-presenting cells and have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, we aimed to develop T cell-based vaccine capable of eliciting potent antitumor therapeutic effects by providing effective costimulatory signals. MATERIALS AND METHODS: Antigenic peptide-loaded T cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax). RESULTS: T cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T cells but not CD4 T cells or natural killer cells. The combination with programmed death-ligand 1 blockade led to potent therapeutic efficacy which exhibited increased tumor-infiltrating CD8 T cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T cells into tumor sites and elicits enhanced therapeutic effects with peptide-based booster immu-nization. CONCLUSION: These results imply that T cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

Comprehensive Analysis of CD4+ T Cell Responses to CMV pp65 Antigen Restricted by Single HLA-DR, -DQ, and -DP Allotype Within an Individual.

Within an individual, six different HLA class II heterodimers are expressed co-dominantly by two alleles of HLA-DR, -DQ, and -DP loci. However, it remained unclear which HLA allotypes were used in T cell responses to a given antigen. For the measurement of the CD4+ T cell responses restricted by a single HLA allotype, we established a panel of artificial antigen-presenting cells (aAPCs) expressing each single HLA allele of 20 HLA-DRB1, 16 HLA-DQ, and 13 HLA-DP alleles. CD4+ T cell responses to cytomegalovirus (CMV) pp65 restricted by single HLA class II allotype defined in 45 healthy donors. The average magnitude of CD4+ T cell responses by HLA-DR allotypes was higher than HLA-DQ and HLA-DP allotypes. CD4+ T cell responses by DRA*01:01/DRB1*04:06, DQA1*01:02/DQB1*06:02, DPA1*02:02/DPB1*05:01 were higher among the other alleles in each HLA-DR, -DQ, and -DP locus. Interestingly, the frequencies of HLA-DR alleles and the positivity of specific allotypes showed an inverse correlation. One allotype within individuals is dominantly used in CD4+ T cell response in 49% of donors, and two allotypes showed that in 7% of donors, and any positive response was detected in 44% of donors. Even if one individual had several dominant alleles, CD4+ T cell responses tended to be restricted by only one of them. Furthermore, CD8+ and CD4+ T cell responses by HLA class I and class II were correlated. Our results demonstrate that the CD4+ T cell preferentially use a few dominant HLA class II allotypes within individuals, similar to CD8+ T cell response to CMV pp65.

GM-CSF Promotes the Expansion and Differentiation of Cord Blood Myeloid-Derived Suppressor Cells, Which Attenuate Xenogeneic Graft-vs.-Host Disease.

Myeloid-derived suppressor cells (MDSCs) are increased in tumor patients. Studies have shown generation of MDSCs from human peripheral blood mononuclear cells (PBMCs) by various cytokine combinations. However, large scale expansion of human MDSCs has not been demonstrated or applied in clinic settings. We investigated which cytokine combinations among GM-CSF/SCF, G-CSF/SCF, or M-CSF/SCF efficiently expand and differentiate human MDSCs following culture CD34+ cells of umbilical cord blood (CB). GM-CSF/SCF showed the greatest expansion of MDSCs. Up to 108 MDSCs (HLA-DRlowCD11b+CD33+) could be produced from 1 unit of CB following 6 weeks of continuous culture. MDSCs produced from culture of CD34+ cells with GM-CSF/SCF for 6 weeks had the greatest suppressive function of T cell proliferation and had the highest expression of immunosuppressive molecules including iNOS, arginase 1 and IDO compared to those differentiated with G-CSF/SCF or M-CSF/SCF. MDSCs secreted IL-10, TGB-ß, and VEGF. The infusion of expanded MDSCs significantly prolonged the survival and decreased the GVHD score in a NSG xenogeneic model of GVHD. Injected MDSCs increased IL-10 and TGF-ß but decreased the level of TNF-α and IL-6 in the serum of treated mice. Notably, FoxP3 expressing regulatory T (Treg) cells were increased while IFN-γ (Th1) and IL-17 (Th17) producing T cells were decreased in the spleen of MDSC treated mice compared to untreated GVHD mice. Our results demonstrate that human MDSCs are generated from CB CD34+ cells using GM-CSF/SCF. These MDSCs exhibited potent immunosuppressive function, suggesting that they are useable as a treatment for inflammatory diseases such as GVHD.

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.

Infusions of Epstein-Barr virus-specific cytotoxic T lymphocytes as post-remission therapy in high-risk post-transplant lymphoproliferative disorder patients: report of two cases.

Conventional therapeutic approaches to post-transplant lymphoproliferative disorder (PTLD) occurring after solid-organ transplantation have shown only limited success in achieving durable response. Key factors driving the pathogenesis of PTLD include Epstein-Barr virus (EBV) reactivation and impaired immune surveillance due to prolonged immune suppression. Thus, EBV-specific cytotoxic T lymphocytes (EBV-CTLs) have emerged as an alternative therapeutic approach for the treatment of EBV-associated PTLD by enhancing EBV-specific immunity. We evaluated the safety and efficacy of EBV latent membrane proteins (LMP)-1- and 2-specific CTLs in two PTLD patients at high risk for relapse. Following diagnosis, patients were initially treated with a combination of chemotherapy and/or radiotherapy. Patients then received a total of eight doses of 2 × 107 EBV-CTLs/m2. Following initial therapy, both patients achieved complete remission confirmed by FDG-PET/CT imaging. Post-remission therapy using adoptive transfer of EBV-CTLs was safe without immediate or late toxicities. Infusion of EBV-CTLs led to an overall reduction in plasma EBV levels in the peripheral blood, which was associated with long-term remission of both patients during a follow-up of more than 65 months. Further prospective studies with larger number of patients will be needed to confirm the role of EBV-CTLs as post-remission therapy in high-risk PTLD.

Comprehensive Analysis of Cytomegalovirus pp65 Antigen-Specific CD8+ T Cell Responses According to Human Leukocyte Antigen Class I Allotypes and Intraindividual Dominance.

To define whether individual human leukocyte antigen (HLA) class I allotypes are used preferentially in human cytomegalovirus (CMV)-specific cytotoxic T lymphocyte responses, CD8+ T cell responses restricted by up to six HLA class I allotypes in an individual were measured in parallel using K562-based artificial antigen-presenting cells expressing both CMV pp65 antigen and one of 32 HLA class I allotypes (7 HLA-A, 14 HLA-B, and 11 HLA-C) present in 50 healthy Korean donors. The CD8+ T cell responses to pp65 in the HLA-C allotypes were lower than responses to those in HLA-A and -B allotypes and there was no difference between the HLA-A and HLA-B loci. HLA-A*02:01, -B*07:02, and -C*08:01 showed the highest magnitude and frequency of immune responses to pp65 at each HLA class I locus. However, HLA-A*02:07, -B*59:01, -B*58:01, -B*15:11, -C*03:02, and -C*02:02 did not show any immune responses. Although each individual has up to six different HLA allotypes, 46% of the donors showed one allotype, 24% showed two allotypes, and 2% showed three allotypes that responded to pp65. Interestingly, the frequencies of HLA-A alleles were significantly correlated with the positivity of specific allotypes. Our results demonstrate that specific HLA class I allotypes are preferentially used in the CD8+ T cell immune response to pp65 and that a hierarchy among HLA class I allotypes is present in an individual.

Antigen Presentation by Individually Transferred HLA Class I Genes in HLA-A, HLA-B, HLA-C Null Human Cell Line Generated Using the Multiplex CRISPR-Cas9 System.

Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients, graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference, zinc finger nuclease, and the CRISPR/Cas9 system. To improve HLA gene editing, we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore, artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.

Simultaneous in vitro generation of CD8 and CD4 T cells specific to three universal tumor associated antigens of WT1, survivin and TERT and adoptive T cell transfer for the treatment of acute myeloid leukemia.

Previously, we found that most patients with acute myeloid leukemia (AML) expressed at least one of the leukemic associated antigens (LAAs) WT1, survivin and TERT, and different combinations of the three LAAs predicted negative clinical outcomes. Multi-tumor antigen-specific T cells were generated to overcome antigenic variation and may be sufficient to maximize antitumoral effects. To generate triple antigen-specific (Tri)-T cells that recognize three LAAs, dendritic cells (DCs) were transfected with three tumor antigen-encoding RNAs. These DCs were used to stimulate both CD8 and CD4 T cells and to overcome the limitation of known human leukocyte antigen-restricted epitopes. The sum of the antigen-specific T cell frequencies was higher in the Tri-T cells than in the T cells that recognized a single antigen. Furthermore, the Tri-T cells were more effective against leukemic blasts that expressed all three LAAs compared with blasts that expressed one or two LAAs, suggesting a proportional correlation between IFN-γ secretion and LAA expression. Engrafted leukemic blasts in the bone marrow of mice significantly decreased in the presence of Tri-T cells. This technique represents an effective immunotherapeutic strategy in AML.

Use of Engineered Exosomes Expressing HLA and Costimulatory Molecules to Generate Antigen-specific CD8+ T Cells for Adoptive Cell Therapy.

Dendritic cell-derived exosomes (DEX) comprise an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to their broad applicability in immunotherapeutic approaches. In previous studies, genetically engineered K562 have been used to generate artificial antigen presenting cells (AAPC). Here, we isolated exosomes from K562 cells (referred to as CoEX-A2s) engineered to express human leukocyte antigen (HLA)-A2 and costimulatory molecules such as CD80, CD83, and 41BBL. CoEX-A2s were capable of stimulating antigen-specific CD8 T cells both directly and indirectly via CoEX-A2 cross-dressed cells. Notably, CoEX-A2s also generated similar levels of HCMV pp65-specific and MART1-specific CD8 T cells as DEX in vitro. The results suggest that these novel exosomes may provide a crucial reagent for generating antigen-specific CD8 T cells for adoptive cell therapies against viral infection and tumors.