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Adoptive transfer of T lymphocytes (ACT) engineered with T-cell receptors (TCRs) of known antitumor specificity is an effective therapeutic strategy. However, a major constraint of ACT is the unpredictable interference of the endogenous TCR α and β chains in pairing of the transduced TCR. This effect reduces the efficacy of the genetically modified primary T cells and carries the risk of generating novel TCR reactivities with unintended functional consequences. Here, we show a powerful approach to overcome these limitations. We engineered TCR α and β chains with mutations encompassing a conserved motif (FXXXFXXS) required to stabilize the pairing of immunoglobulin heavy chain transmembrane domains. Molecular modeling supported the preferential pairing of mutated TCR and impaired pairing between mutated and wild-type TCRs. Expression of the mutated TCR was similar to wild type and conferred the expected specificity. Fluorescence resonance energy transfer analysis in mouse splenocytes transduced with mutated or wild-type TCRs showed a higher proximity of the former over the latter. Importantly, we show that mutated TCRs effectively outcompete endogenous TCRs and improve in vitro antitumor cytotoxicity when expressed in ex vivo isolated human T cells. This approach should contribute to improving current protocols of anticancer immunetherapy protocols.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

Article history:Received 27 July 2011Received in revised form 6 September 2011Accepted 7 September 2011Keywords:Antarctic skateCartilaginous fish immunoglobulin MCμ gene diversityExtra-cysteines Immunoglobulins (Ig) of Chondroichthyes have been extensively studied in sharks; in contrast, in skatesinvestigations on Ig remain scarce and fragmentary despite the high occurrence of skates in all of themajor oceans of the world. To focus on Rajidae Igμ, the most abundant heavy chain isotype, we have chosenthe Antarctic species Bathyraja eatonii, Bathyraja albomaculata, Bathyraja brachyurops, and Amblyrajageorgiana which live at high latitudes in the Southern Ocean, and at very low temperatures. We preparedmRNA from the spleen of individuals of each species and performed RT-PCR experiments using twooligonucleotides designed on the alignment of various elasmobranch Igμ heavy chain sequences availablein GenBank. The PCR products, about 1400-nt long, were cloned and sequenced. Nucleotide sequence identi-ties calculated for the constant region domains ranged from 88.5% to 97.5% between species, and from 91.1%to 99.7% within species. In a distance tree, including also Raja erinacea sequences, two major branches wereobtained, one containing Arhynchobatinae sequences, the other one Rajinae sequences. Four presumptive Dgene segments were identified in the region of the VH/D/JH recombination; two different D segments wereoften found in the same sequence. Moreover, 5–15 genomic fragments of different lengths, carrying thegene locus encoding Igμ chain were revealed by Southern blotting analysis. B. eatonii amino acid sequenceswere analyzed for the positional diversity by Shannon entropy analysis, showing CH4 as the most conserveddomain, and CH3 as the most variable one. B. eatonii CDR3 region length varied between 11 and 15 aminoacid residues; the mean length (13.4 aa) was greater than that of Leucoraja eglanteria sequences (7.7 aa).An alignment of representative sequences of Antarctic species and R. erinacea showed that more cysteineresidues not involved in the intradomain disulfide bridges were present in Antarctic species.© 2011 Elsevier B.V. All rights reserved.

Filamentous bacteriophage fd particles delivering antigenic determinants via DEC‐205 (fdsc‐αDEC) represent a powerful delivery system that induces CD8 + T‐cell responses even when administered in the absence of adjuvants or maturation stimuli for dendritic cells. In order to investigate the mechanisms of this activity, RNA‐Sequencing of fd‐pulsed dendritic cells was performed. A significant differential expression of genes involved in innate immunity, co‐stimulation and cytokine production was observed. In agreement with these findings, we demonstrate that induction of proinflammatory cytokines and type I interferon by fdsc‐αDEC was MYD88 mediated and TLR9 dependent. We also found that fdsc‐αDEC is delivered into LAMP‐1‐positive compartments and co‐localizes with TLR9. Thus, phage particles containing a single‐strand DNA genome rich in CpG motifs delivered via DEC‐205 are able to intercept and trigger the active TLR9 innate immune receptor into late endosome/lysosomes and to enhance the immunogenicity of the displayed antigenic determinants. These findings make fd bacteriophage a valuable tool for immunization without administering exogenous adjuvants.