Targeting of the WT191-138 fragment to human dendritic cells improves leukemia-specific T-cell responses providing an alternative approach to WT1-based vaccination.

Due to its immunogenicity and overexpression concomitant with leukemia progression, Wilms tumor protein 1 (WT1) is of particular interest for immunotherapy of AML relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, WT1-specific T-cell responses have mainly been induced by vaccination with peptides presented by certain HLA alleles. However, this approach is still not widely applicable in clinical practice due to common limitations of HLA restriction. Dendritic cell (DC) vaccines electroporated with mRNA encoding full-length protein have also been tested for generating WT1-derived peptides for presentation to T-cells. Alternatively, an efficient and broad WT1 peptide presentation could be elicited by triggering receptor-mediated protein endocytosis of DCs. Therefore, we developed antibody fusion proteins consisting of an antibody specific for the DEC205 endocytic receptor on human DCs and various fragments of WT1 as DC-targeting recombinant WT1 vaccines (anti-hDEC205-WT1). Of all anti-hDEC205-WT1 fusion proteins designed for overcoming insufficient expression, anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were identified in good yields. The anti-hDEC205-WT191-138 was capable of directly inducing ex vivo T-cell responses by co-incubation of the fusion protein-loaded monocyte-derived mature DCs and autologous T-cells of either healthy or HSCT individuals. Furthermore, the DC-targeted WT191-138-induced specific T-cells showed a strong cytotoxic activity by lysing WT1-overexpressing THP-1 leukemia cells in vitro while sparing WT1-negative hematopoietic cells. In conclusion, our approach identifies four WT1 peptide-antibody fusion proteins with sufficient production and introduces an alternative vaccine that could be easily translated into clinical practice to improve WT1-directed antileukemia immune responses after allo-HSCT.

Heterogeneity of Specific CD4+ and CD8+ T Cells Stimulated by CMV pp65 and IE1 Antigens.

Characterization of human cytomegalovirus-specific T cells (CMV-T) is of critical importance for their potential use in adoptive immunotherapy after allogeneic hematopoietic stem cell transplantation. Background frequencies of CMV-T in peripheral blood mononuclear cells (PBMCs) of CMV-seropositive healthy subjects are usually very low, hence the requirement for prolonged culture time and multiple stimulations to expand them. The evaluation of the end-culture specificity and composition has sometimes been neglected or difficult to assess in these settings. We explored the identity and the functionality of pp65-specific and IE1-specific T cells, enriched in short-term cultures from PBMCs. Antigen-specific T cells were further isolated by IFN-γ capture system and/or CD154 microbeads. Frequency of IE1-specific cytotoxic T cells in PBMCs secreting IFN-γ was higher compared with the pp65-specific one, whereas the latter cell types showed a higher median CD107a degranulation. Cell viability, rate of CMV-T increase, and multicytokine secretion profile after epitope-specific short-term cultures were heterogenous. T cells were mainly of late effector stages but they significantly dropped off upon CMV rechallenge with peptide pools. In parallel, CMV-T expansion was accompanied by a significant increase of cytotoxic naive/memory stem cells (CTLs), whereas the CD4 counterpart significantly increased only upon stimulation with IE1. Outcome was variable and showed donor and epitope dependency. Differences in human leukocyte antigen and epitope dominance and variability in the relative number of CD3 effector cells and IFN-γ/CD154 expression among healthy donors could reflect the observed individual CMV-specific cellular immunity. This heterogeneity raises points to be considered when approaching adoptive immunotherapy.

Transmission of oxLDL-derived lipid peroxide radicals into membranes of vascular cells is the main inducer of oxLDL-mediated oxidative stress.

Oxidatively modified LDL is generally accepted to be an important elicitor of pro-mitotic, pro-inflammatory, and atherogenic effects in vascular cells. The uptake of oxLDL and concomitant activation of the O(2)*-producing NAD(P)H oxidase and/or oxLDL as a self-contained emitter of O(2)* are believed to trigger these malfunctions. The following observations allowed reinvestigating the mode of oxLDL-induced stress: (1) we observed that artery smooth muscle primary cells internalize fluorescently labelled oxidized or acetylated LDL considerably less efficient than endothelial cells. (2) Both types of cells, however, displayed an oxLDL concentration dependent level of oxidative stress as monitored by the oxidation of carboxy-H2DCFDA to fluorescent carboxy-DCF. A dose dependent decrease of dihydroethidine oxidation to oxyethidine implied an oxLDL-induced depletion of the cellular energy pool. The release of O(2)* by exogenous oxLDL, as postulated above, did not sufficiently explain intracellular stress because the fluorescence was only marginally blocked by antioxidative enzymes (SOD, catalase) or substances (L-NAME, DMSO, DMHP, DMTU). We were able to reveal a third mode of oxLDL-induced stress by showing with the help of a fluorescent, oxidizable lipid analogue (BODIPY 581/591 C(11)) that oxLDL-derived lipid peroxides and radicals migrate into cellular membranes giving rise to a chronic inoculation of the vascular cells with oxidative chain reactions. The novel data may help to design adequate therapeutic strategies against oxLDL-induced cardiovascular diseases.