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.

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.

Triple costimulation via CD80, 4-1BB, and CD83 ligand elicits the long-term growth of Vγ9Vδ2 T cells in low levels of IL-2.

Human γδ T cells play important roles in the regulation of infection and cancer. To understand the roles of costimulatory signals in activation and expansion ex vivo, Vγ9Vδ2 T cells were grown with artificial APCs that express CD83, 4-1BB ligand, and/or CD32, which allowed a loading of αCD3 and αCD28 antibodies. The costimulatory signals through CD80, 4-1BB, and CD83 ligand in low levels of IL-2 triggered an explosive ex vivo proliferation of Vγ9Vδ2 T cells capable of secreting high levels of IL-2, IFN-γ, and TNF-α. Moreover, the triple-costimulatory signals cause augmented cell viabilities for long-term growth of Vγ9Vδ2 T cells, resulting in phenotypic changes to CD27(-)CD45RA(+) effector memory-like cells. Notably, we observed that CD83 ligand signaling is crucial to promote ex vivo expansion, survival, and cytolytic effector functions of Vγ9Vδ2 T cells. In contrast, 4-1BB signaling is moderately important in up-regulating surface molecules on Vγ9Vδ2 T cells. Consequently, γδ T cells stimulated in the presence of triple-costimulatory signals have diverse cytolytic effector molecules, including perforin, granzyme A, granzyme B, and Fas ligand, eliciting potent cytolytic activities against tumor cells. Overall, our results provide insights into the roles of costimulatory signals in manufacturing long-lived and fully functional Vγ9Vδ2 T cells that could be useful against cancers.

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs.

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8(+) and CD4(+) T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8(+) and CD4(+) T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4(+) T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4(+) T cells was significantly correlated with that of CD8(+) T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4(+) T cell responses showed more significant changes than CD8(+) T cell responses. CD8(+) and CD4(+) T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4(+) T cells secreted significantly higher cytokine levels than did CD8(+) T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.