Sébastien Wieckowski

Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type

The development of nanomaterials for biomedical and biotechnological applications is an area of research that holds great promise and intense interest1, and carbon-based nanostructures in particular, such as carbon nanotubes (CNTs), are attracting an increasing level of attention2,3. One of the key advantages that CNTs offer is the possibility of effectively crossing biological barriers, which would allow their use in the delivery of therapeutically active molecules. Our laboratories have been investigating the use of CNTs in biomedical applications, and in particular as nanovectors for therapeutic agent delivery4,5,6,7,8. The interaction between cells and CNTs is a critical issue that will determine any future biological application of such structures. Here we show that various types of functionalized carbon nanotubes (f-CNTs) exhibit a capacity to be taken up by a wide range of cells and can intracellularly traffic through different cellular barriers.

Fine Structural Variations of αβTCRs Selected by Vaccination with Natural versus Altered Self-Antigen in Melanoma Patients

Immunotherapy of cancer is often performed with altered “analog” peptide Ags optimized for HLA class I binding, resulting in enhanced immunogenicity, but the induced T cell responses require further evaluation. Recently, we demonstrated fine specificity differences and enhanced recognition of naturally presented Ag by T cells after vaccination with natural Melan-A/MART-1 peptide, as compared with analog peptide. In this study, we compared the TCR primary structures of 1489 HLA-A*0201/Melan-A26–35-specific CD8 T cells derived from both cohorts of patients. Although a strong preference for TRAV12-2 segment usage was present in nearly all patients, usage of particular TRAJ gene segments and CDR3α composition differed slightly after vaccination with natural vs analog peptide. Moreover, TCR β-chain repertoires were broader after natural than analog peptide vaccination. In all patients, we observed a marked conservation of the CDR3β amino acid composition with recurrent sequences centered on a glycyl-leucyl/valyl/alanyl-glycyl motif. In contrast to viral-specific TCR repertoires, such “public” motifs were primarily expressed by nondominant T cell clonotypes, which contrasted with “private” CDR3β signatures frequently found in T cell clonotypes that dominated repertoires of individual patients. Interestingly, no differences in functional avidity were observed between public and private T cell clonotypes. Collectively, our data indicate that T cell repertoires generated against natural or analog Melan-A peptide exhibited slightly distinct but otherwise overlapping and structurally conserved TCR features, suggesting that the differences in binding affinity/avidity of TCRs toward pMHC observed in the two cohorts of patients are caused by subtle structural TCR variations.

Cutting edge: small molecule CD40 ligand mimetics promote control of parasitemia and enhance T cells producing IFN-gamma during experimental Trypanosoma cruzi infection.

Host resistance to Trypanosoma cruzi infection depends on a type 1 response characterized by a strong production of IL-12 and IFN-γ. Amplifying this response through CD40 triggering results in control of parasitemia. Two newly synthesized molecules (<3 kDa) mimicking trimeric CD40L (mini CD40Ls-1 and -2) bind to CD40, activate murine dendritic cells, and elicit IL-12 production. Wild-type but not CD40 knockout mice exhibited a sharp decrease of parasitemia and mortality when inoculated with T. cruzi mixed with miniCD40Ls. Moreover, the immunosuppression induced by T. cruzi infection was impaired in mice treated with miniCD40Ls, as shown by proliferation of splenic lymphocytes, percentage of CD8+ T cells, and IFN-γ production. Mice surviving T. cruzi infection in the presence of miniCD40L-1 were immunized against a challenge infection. Our results indicate that CD40L mimetics are effective in vivo and promote the control of T. cruzi infection by overcoming the immunosuppression usually induced by the parasites.

Solid-phase synthesis of CD40L mimetics

The C3-symmetric molecule has been previously shown to mimic CD40 ligand (CD40L) homotrimers and to display effector functions. This molecule consists of a cyclic hexapeptide core containing the repetition of the D-Ala-L-Lys motif. The side chains of the lysine residues have been modified by appending the CD40L-derived sequence 143Lys-Gly-Tyr-Tyr146 via a 6-aminohexanoic acid residue as a spacer. The present report describes a general solid-phase synthesis approach to and related trimeric architectures. In addition, their CD40 binding properties as well as their effector functions have been evaluated.

Immunomodulatory consequences of ODN CpG-polycation complexes.

Immunostimulatory ODN CpGs have extensively been tested as adjuvants and immunotherapeutics and hold a lot of promise for human use. In our studies we took advantage of their negative charge to study their biological activities after being complexed with carbon nanotubes, a novel vector for vaccine delivery and Tat protein of HIV, a target protein for therapeutic or prophylactic intervention. In the case of carbon nanotubes, ODN CpGs were able to form stable complexes based on charge interaction and exert increased immunostimulatory activity in vitro. With regard to the Tat protein, ODN CpGs were shown to bind effectively through the basic domain of the protein representing residues 44-61. Moreover, using surface Plasmon Resonance Technology and an in vitro cellular system, ODN CpGs were shown to inhibit the interaction of Tat protein with the transactivation responsive element, a bulged RNA hairpin structure. However, when ODN CpGs were complexed with Tat they readily increased the apoptotic properties of this protein as studied in CD3-stimulated Jurkat cells. Overall, our findings together with published data support the view that for harnessing the beneficial effects of ODN CpGs a careful consideration has to be given depending on the target intervention.