Aberrant expression of regulatory receptors programmed death-1 (PD-1) and B-and T-lymphocyte attenuator (BTLA) is linked with dysregulation and exhaustion of T lymphocytes during chronic human immunodeficiency virus type 1 (HIV-1) infection; however, less is known about whether a similar process impacts B-lymphocyte function during HIV-1 infection. We reasoned that disruption of the peripheral B cell compartment might be associated with decreased neutralizing antibody activity. Expression of markers that indicate dysregulation (BTLA and PD-1), immune activation (CD95), and proliferation (Ki-67) was evaluated in B cells from HIV-1-infected viremic and aviremic subjects and healthy subjects, in conjunction with immunoglobulin production and CD4 T cell count. Viral load and cross-clade neutralizing activity in plasma from viremic subjects were also assessed. Dysregulation of B lymphocytes was indicated by a marked disruption of peripheral B cell subsets, increased levels of PD-1 expression, and decreased levels of BTLA expression in viremic subjects compared to aviremic subjects and healthy controls. PD-1 and BTLA were correlated in a divergent fashion with immune activation, CD4 T cell count, and the total plasma IgG level, a functional correlate of B cell dysfunction. Within viremic subjects, the total IgG level correlated directly with cross-clade neutralizing activity in plasma. The findings demonstrate that even in chronically infected subjects in which B lymphocytes display multiple indications of dysfunction, antibodies that mediate cross-clade neutralization breadth continue to circulate in plasma.
We investigated the use of cytotoxic T-lymphocyte (CTL) epitopes in peptide immunotherapy for glioblastoma. Three peptides (ERBB2, BIRC5 and CD99) were selected based on their peptide-T2 cell binding affinities and combined in a multipeptide cocktail or a branched multipeptide synthesized with mini-polyethylene glycol spacers. Dendritic cells (DCs) pulsed with the multipeptide cocktail or branched multipeptide were compared based on their immunophenotype and cytokine secretion. FACS analysis of alpha-type 1 polarized dendritic cells (αDC1s) revealed that both groups highly expressed CD80, CD83 and CD86, indicating that both treatments efficiently generated mature αDC1s with the expected phenotype. Production of IL-12p70, IL-12p40 and IL-10 also increased upon αDC1 maturation in both groups. CTLs stimulated by either αDC1 group (“DC-CTLs”) included numerous IFN-γ-secreting cells against T2 cells loaded with the corresponding multipeptides. Large numbers of IFN-γ-secreting cells were observed when human glioblastoma cell lines and primary cells were treated with multipeptide-pulsed DC-CTLs. Both multipeptide-pulsed DC-CTL groups exhibited cytotoxic activity of 40-60% against the U251 cell line and 60-80% against primary cells. Branched multipeptide from ERBB2, BIRC5 and CD99 stably bound with T2 cells, and its cytotoxicity toward target cells was similar to that of the multipeptide cocktail. Thus, branched multipeptides could be promising candidates for immunotherapeutic glioblastoma treatment.
Various combination treatments have been considered to attain the effective therapy threshold by combining independent antitumor mechanisms against the heterogeneous characteristics of tumor cells in malignant brain tumors. In this study, the natural killer (NK) cells associated with bevacizumab (Bev) plus irinotecan (Iri) against glioblastoma multiforme (GBM) were investigated. For the experimental design, NK cells were expanded and activated by K562 cells expressing the OX40 ligand and membrane-bound IL-18 and IL-21. The effects of Bev and Iri on the proliferation and NK ligand expression of GBM cells were evaluated through MTT assay and flow cytometry. The cytotoxic effects of NK cells against Bev plus Iri-treated GBM cells were also predicted via the LDH assay in vitro. The therapeutic effect of different injected NK cell routes and numbers combined with the different doses of Bev and Iri was confirmed according to tumor size and survival in the subcutaneous (s.c) and intracranial (i.c) U87 xenograft NOD/SCID IL-12Rγnull mouse model. The presence of injected-NK cells in tumors was detected using flow cytometry and immunohistochemistry ex vivo. As a result, Iri was found to affect the proliferation and NK ligand expression of GBM cells, while Bev did not cause differences in these cellular processes. However, the administration of Bev modulated Iri efficacy in the i.c U87 mouse model. NK cells significantly enhanced the cytotoxic effects against Bev plus Iri-treated GBM cells in vitro. Although the intravenous (IV) injection of NK cells in combination with Bev plus Iri significantly reduced the tumor volume in the s.c U87 mouse model, only the direct intratumorally (IT) injection of NK cells in combination with Bev plus Iri elicited delayed tumor growth in the i.c U87 mouse model. Tumor-infiltrating NK cells were detected after IV injection of NK cells in both s.c and i.c U87 mouse models. In conclusion, the potential therapeutic effect of NK cells combined with Bev plus Iri against GBM cells was limited in this study. Accordingly, further research is required to improve the accessibility and strength of NK cell function in this combination treatment.
The promising immunotherapy effects of a multiple antigenic peptide on glioblastoma (GBM) in a previous study encourage the use of adjuvants to enhance its therapeutic efficacy. Among adjuvants, pan HLA-DR-binding epitope (PADRE) and anti-programmed cell death protein 1 (anti-PD1) have potentially been tested for cancer immunotherapy. Therefore, here we evaluated the ability of PADRE and anti-PD1 to enhance the function of the branched multipeptide against GBM. The potential utility of tumor-associated antigens (ErbB-2 and WT-1) targeting GBM with HLA-A24 was confirmed and a branched multipeptide was constructed from these antigens. The effects of the branched multipeptide and PADRE on immunophenotyping and polarized Th cytokine production in dendritic cells were clarified. The expression of PD1 on T cells and PDL1 on GBM cells was also investigated. The interferon-γ enzyme-linked immunospot and lactate dehydrogenase release assays were performed to determine the function of GBM peptide antigen-specific cytotoxic T cells against GBM cells. Overall, this study showed that both ErbB-2 and WT-1 are potential candidates for branched multipeptide construction. The branched multipeptide and PADRE enhanced the expression of major histocompatibility complex and co-stimulatory molecules and the production of polarized Th1 cytokines in dendritic cells. The increase in the number of interferon-γ + effector T cells was consistent with the increase in the percentage specific lysis of GBM target cells by GBM peptide antigen-specific cytotoxic T cells in the presence of the branched multipeptide, PADRE, and anti-PD1. Our study suggests the combination of branched multipeptide and adjuvants such as PADRE and anti-PD1 can potentially enhance the effects of immunotherapy for GBM treatment.
Emerging data have suggested that single short peptides have limited success as a cancer vaccine; however, extending the short peptides into longer multi-epitope peptides overcame the immune tolerance and induced an immune response. Moreover, the combination of adjuvants such as lenalidomide and anti-programmed cell death protein 1 (PD1) with a peptide vaccine showed potential vaccine effects in previous studies. Therefore, the effects of a long multi-epitope peptide vaccine in combination with lenalidomide and anti-PD1 were analyzed in this study. Long multi-epitope peptides from two MHCI peptides (BIRC597-104 and EphA2682-689) and the pan-human leukocyte antigen-DR isotype (HLA-DR) binding epitope (PADRE) were synthesized. The therapeutic effects of long multi-epitope peptides in combination with lenalidomide and anti-PD1 were confirmed in the murine GL261 intracranial glioma model. Immune cells’ distribution and responses to the long multi-epitope peptides in combination with these adjuvants were also estimated in the spleens, lymph nodes, and tumor tissues. The difference between long multi-epitope peptides and a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 was also clarified. As a result, long multi-epitope peptides combined with lenalidomide and anti-PD1 prolonged the survival of mice according to the suppression of tumor growth in an intracranial mouse model. While long multi-epitope peptides combined with these adjuvants enhanced the percentages of activated and memory effector CD8+ T cells, the increase in percentages of regulatory T cells (Tregs) was observed in a cocktail of multi-epitope peptides combined with lenalidomide and anti-PD1 group in the tumors. Long multi-epitope peptides combined with these adjuvants also enhanced the function of immune cells according to the enhanced pro-inflammatory cytokines and cytotoxicity against GL261 cells in ex vivo. In conclusion, long multi-epitope peptides composed of MHCI peptides, BIRC5 and EphA2, and the MHCII peptide, PADRE, in combination with lenalidomide and anti-PD1 has the potential to improve the therapeutic effects of a vaccine against GBM.
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