Exposure-dependent Ag+ release from silver nanoparticles and its complexation in AgS2 sites in primary murine macrophages.

Silver nanoparticle (AgNP) toxicity is related to their dissolution in biological environments and to the binding of the released Ag(+) ions in cellulo; the chemical environment of recombined Ag(+) ions is responsible for their toxicological outcome, moreover it is indicative of the cellular response to AgNP exposure, and can therefore shed light on the mechanisms governing AgNP toxicity. This study probes the chemistry of Ag species in primary murine macrophages exposed to AgNPs by making use of X-ray Absorption Fine Structure spectroscopy under cryogenic conditions: the linear combination analysis of the near-edge region of the spectra provides the fraction of Ag(+) ions released from the AgNPs under a given exposure condition and highlights their complexation with thiolate groups; the ab initio modelling of the extended spectra allows measuring the Ag-S bond length in cellulo. Dissolution rates depend on the exposure scenario, chronicity leading to higher Ag(+) release than acute exposure; Ag-S bond lengths are 2.41 ± 0.03 Å and 2.38 ± 0.01 Å in acute and chronic exposure respectively, compatible with digonal AgS2 coordination. Glutathione is identified as the most likely putative ligand for Ag(+). The proposed method offers a scope for the investigation of metallic nanoparticle dissolution and recombination in cellular models.

Preferential ADV-AJ association during recombination in the mouse T-cell receptor alpha/delta locus.

The gene coding for a T-cell receptor (TCR) alpha chain is assembled from variable (ADV) and joining (AJ) genes located on Chromosome 14. Each of the 90 ADV genes can rearrange with any one of the 61 AJ genes. We have previously demonstrated that ADV and AJ gene segment use evolves with time, with a progressive opening of ADV and AJ regions of the locus. To define the rules governing the use of AJ genes by ADV genes belonging to one family, we carried out a detailed analysis of 268 combinations of ADV2 BALB/c transcripts. We found that the different ADV2 members use different sets of AJ genes depending on their location within the ADV locus: ADV2S7 (the most AJ proximal ADV2 member) rearranges mainly with the AJ genes located close to the TEA element, whereas 50% of the sequences for ADV2S8, which is distal to the AJ locus, use the most distal AJ genes. ADV2S5, an ADV2 member located in the middle of the ADV locus, is associated with a wider set of AJ genes, located in the center of the AJ locus. Taken together, our results indicate that, in addition to the progressive opening of the ADV and AJ loci, the chromosomal location of ADV and AJ genes is a factor affecting AJ use in BALB/c mice.

Tetanus toxin L chain is processed by major histocompatibility complex class I and class II pathways and recognized by CD8+ or CD4+ T lymphocytes.

Tetanus toxin (TeNT) is a heterodimeric protein antigen, whose light chain (L) is translocated in the cytosol of neuronal target cells specifically to cleave its substrates, vesicle-associated membrane protein-2 (VAMP-2, or synaptobrevin) or cellubrevin. We report that the L chain behaves as a nominal antigen recognized by specific T-cell clones upon either class I- or II-restricted presentation. Three types of responses are observed: (i) a TeNT- and L-specific CD8+ T-cell response, that can be inhibited in a dose-dependent manner by the proteasome inhibitor clasto-Lactacystin beta-lactone; (ii) a CD4+ T-cell response specific for L but not TeNT, with recognition of a determinant processed in a chloroquine-sensitive and brefeldin A-resistant compartment; (iii) a CD4+ T-cell response against both L and TeNT, with processing in a brefeldin A-sensitive compartment. The L chain processing was investigated in U937 cells by internalization and localization of L chain by separation of the cell content by differential centrifugation experiments. After incubation with TeNT or L chain in the presence of H chain, the L chain was predominantly distributed in the cytosolic fraction, whereas incubation with L alone led to localization in a lysosome/membrane fraction. The distribution of the TeNT L chain in both cytosolic and endocytic compartments of the antigen-presenting cell accounted for its processing by both class I and class II pathways. Furthermore, an epitope overlapping with the zinc-binding region was recognized by CD4+ and CD8+ T cells.

Pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 superantigen.

Superantigens (SAg) are proteins of bacterial or viral origin able to activate T cells by forming a trimolecular complex with both MHC class II molecules and the T cell receptor (TCR), leading to clonal deletion of reactive T cells in the thymus. SAg interact with the TCR through the beta chain variable region (Vbeta), but the TCR alpha chain has been shown to have an influence on the T cell reactivity. We have investigated here the role of the TCR alpha chain in the modulation of T cell reactivity to Mtv-7 SAg by comparing the peripheral usage of Valpha2 in Vbeta6(+) (SAg-reactive) and Vbeta8.2(+) (SAg non-reactive) T cells, in either BALB/D2 (Mtv-7(+)) or BALB/c (Mtv-7(-)) mice. The results show, first, that pairing of Vbeta6 with certain Valpha2 family members prevents T cell deletion by Mtv-7 SAg. Second, there is a strikingly different distribution of the Valpha2 family members in CD4 and CD8 populations of Vbeta6 but not of Vbeta8.2 T cells, irrespective of the presence of Mtv-7 SAg. Third, the alpha chain may play a role in the overall stability of the TCR/SAg/MHC complex. Taken together, these results suggest that the Valpha domain contributes to the selective process by its role in the TCR reactivity to SAg/MHC class II complexes, most likely by influencing the orientation of the Vbeta domain in the TCR alphabeta heterodimer.

Differential chronology of TCRADV2 gene use by alpha and delta chains of the mouse TCR.

The genes coding for TCR alpha and delta chains share the same genetic locus (TCRA/D). The rules governing the utilization of a V gene with the alpha and delta chains have not been established. More specifically, it is not known whether the position of a gene within the locus influences its utilization in alpha and delta TCR. To elucidate these points, we mapped ADV2 genes in the TCRA/D locus of BALB/c mice and analyzed their utilization in TCR alpha and delta transcripts from thymi isolated from mice of different ages. Our results show that all ADV2 genes can be used by the two chains, but with strikingly different patterns. Moreover, ADV2 utilization by the alpha chain proceeds in successive concentric waves during development, suggesting a progressive regulation of gene accessibility and utilization. These results support independent control of TCRA and TCRD gene assembly.