Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses.

Adoptive T-cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, which introduces antigen-specific T-cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T-cell repertoires. Studies have suggested that use of higher-affinity TCRs against class I major histocompatibility complex antigens could drive the activity of both CD4(+) and CD8(+) T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here, we describe a high-throughput platform of 'reverse biochemistry' whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes (TILs) or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8(+) T cells expressing the highest-affinity TCR variants were deleted in both the TIL population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4(+) T cells in the tumor, suggesting they had a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR-binding properties that promote peripheral T-cell survival and tumor elimination.

Electrocatalytic responses at mediator modified electrodes with several cyclic step and cyclic sweep potential techniques. Application to the oxidation of ascorbate at a ferrocene-monolayer modified gold electrode.

Analytical expressions for the current-potential and charge-potential curves of an electrocatalytic process taking place at redox mediator modified electrodes corresponding to the application of cyclic staircase or cyclic sweep potentials are presented. Simple equations for cyclic voltammetry and cyclic voltcoulometry curves have been also deduced for the usual experimental case corresponding to high catalytic rate constants, showing that whereas the current reach an stationary behavior, the charge always depend on time. The value of the rate constant of the chemical step can be easily determined from the plateau of the voltammogram or from the linear anodic region of the charge potential curves. The theoretical predictions have been tested with the electrocatalytic oxidation of ascorbate mediated by a mixed 6-(ferrocenyl)hexanethiol-butanethiol monolayer at a disk gold electrode, with an excellent agreement between theory and experiments. A catalytic rate constant 3440 M(-1) s(-1) has been obtained, which is higher than those previously reported for this system with longer alkanethiol chains.

Confinement-induced order of tethered alkyl chains at the water/vapor interface.

Packing of tethered alkyl chains in Langmuir monolayers of a hairy-rod polypeptide poly[gamma-4-(n-hexadecyloxy)benzyl alpha,L-glutamate] on water has been studied by x-ray scattering measurements at room temperature. The rods lie parallel to the surface while the alkyl side chains segregate toward the vapor. Results indicate that the herringbone order of the alkyl chains is established initially by one-dimensionally confined chains between aligned rods and grows laterally with compression.