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PDF file - 277KB, Phenotypic analysis of CMV pp65 RNA-pulsed DCs.

PDF file - 69KB, Cytokine secretion analysis of CMV pp65-specific T cells co-cultured with primary GBM tumor cells.

PDF file - 72KB, Phenotype of in vitro stimulated T cells.

Purpose: Despite aggressive conventional therapy, glioblastoma (GBM) remains uniformly lethal. Immunotherapy, in which the immune system is harnessed to specifically attack malignant cells, offers a treatment option with less toxicity. The expression of cytomegalovirus (CMV) antigens in GBM presents a unique opportunity to target these viral proteins for tumor immunotherapy. Although the presence of CMV within malignant gliomas has been confirmed by several laboratories, its relevance as an immunologic target in GBM has yet to be established. The objective of this study was to explore whether T cells stimulated by CMV pp65 RNA-transfected dendritic cells (DC) target and eliminate autologous GBM tumor cells in an antigen-specific manner.Experimental Design: T cells from patients with GBM were stimulated with autologous DCs pulsed with CMV pp65 RNA, and the function of the effector CMV pp65-specific T cells was measured.Results: In this study, we demonstrate the ability to elicit CMV pp65-specific immune responses in vitro using RNA-pulsed autologous DCs generated from patients with newly diagnosed GBM. Importantly, CMV pp65-specific T cells lyse autologous, primary GBM tumor cells in an antigen-specific manner. Moreover, T cells expanded in vitro using DCs pulsed with total tumor RNA demonstrated a 10- to 20-fold expansion of CMV pp65-specific T cells as assessed by tetramer analysis and recognition and killing of CMV pp65-expressing target cells.Conclusion: These data collectively demonstrate that CMV-specific T cells can effectively target glioblastoma tumor cells for immunologic killing and support the rationale for the development of CMV-directed immunotherapy in patients with GBM. Clin Cancer Res; 20(10); 2684–94. ©2014 AACR.

PDF file - 65KB, Analysis of maturation markers in dendritic cells pulsed with RNA and cultured in maturation cytokine cocktail.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

To test the safety and immunogenicity of a high-titered preparation of ALVAC-HIV vCP205 in both high-risk and low-risk persons and to evaluate variations in dosing schedule, we conducted a multicenter, randomized, double-blind trial of this vector in combination with recombinant subunit gp120 in 150 HIV-1-seronegative volunteers. The high-titered ALVAC vaccine was well tolerated; adverse events were minimal and not influenced by dosing. At day 728, the cumulative probability of a cytotoxic T-lymphocyte (CTL) response was 76% (95% confidence interval [CI]: 64%-89%) among volunteers receiving vaccine, and the net amount attributable to vaccination was 50% (CI: 16%; 74%). The net probability of a repeated positive CTL response by day 728 was 50% (CI: 21%; 64%). There was a significant difference in CTL response at day 182 between volunteers who had received four doses versus three doses of vCP205 (42% vs. 24%, p =.052). The CTL response was similar in high-risk volunteers and vaccinia-naive volunteers compared with vaccinia-immune volunteers. Neutralizing antibody responses were detected in 95% of vaccinees at day 287, with higher geometric mean titers in recipients of sequential versus simultaneous dosing of the two vaccines and in vaccinia-naive volunteers. This high-titered preparation of ALVAC-HIV vCP205 in combination with gp120 was safe and immunogenic in a diverse group of HIV-1-seronegative volunteers.

CD4 T cells are vital in regulating CD8 T cell responses during chronic viral infections. Notably, the hallmark of HIV infection is the severe loss of CD4 T cells resulting in defective CD8 T cell responses. One consequence of CD4 depletion following HIV infection is the loss of interleukin-21 (IL-21) production by the CD4 T cells, which has been demonstrated to be a critical factor in promoting viral control in murine studies. However, it is unknown whether IL-21 can rescue the exhausted CD8 T cells in chronically infected HIV+ patients. In this study we build upon novel murine studies to demonstrate a key role for IL-21 in improving CD8 T cell responses in PBMC from chronically infected HIV patients. To determine whether IL-21 can rescue exhausted CD8 T cells, PBMCs from HIV patients with viral loads > 10,000 RNA copies/mL were incubated with IL-21. Following re-stimulation with overlapping Gag-peptide, IL-21 enhanced the production of IFN-γ by CD8 T cells although no effect was observed on IL-2 or TNF-α production. In addition, an increase in the central memory (CD45RO+CD27+) population was observed with IL-21 culture along with higher MFI of Bcl-6 levels. Interestingly, IL-21 cultured CD8 T cells expressed higher MFI of PD-1 even though CD8 T cell function was enhanced. Significantly, these results provide an approach in which IL-21 can be potentially used to enhance CD8 T cell responses in chronically infected HIV patients, whose CD4 counts are low or defective.

The principal neutralizing determinant (PND) of human immunodeficiency virus HIV-1 is part of a disulfide bridged loop in the third variable region of the external envelope protein, gp120. Analysis of the amino acid sequences of this domain from 245 different HIV-1 isolates revealed that the PND is less variable than thought originally. Conservation to better than 80 percent of the amino acids in 9 out of 14 positions in the central portion of the PND and the occurrence of particular oligopeptide sequences in a majority of the isolates suggest that there are constraints on PND variability. One constraining influence may be the structural motif (beta strand--type II beta turn--beta strand--alpha helix) predicted for the consensus PND sequence by a neural network approach. Isolates with a PND similar to the commonly investigated human T cell lymphoma virus IIIB (HTLV-IIIB) and LAV-1 (BRU) strains were rare, and only 14 percent of sera from 86 randomly selected HIV-1 seropositive donors contained antibodies that recognized the PND of these virus isolates. In contrast, over 65 percent of these sera reacted with peptides containing more common PND sequences. These results suggest that HIV vaccine immunogens chosen because of their similarity to the consensus PND sequence and structure are likely to induce antibodies that neutralize a majority of HIV-1 isolates.