Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis

Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)–matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4+ T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19L-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19L-specific CD4+ T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8+ mHag-specific T cells. They also lysed CD19L-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19L-derived HLA class II–restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings

Polarization Portraits of Single Multichromophoric Systems: Visualizing Conformation and Energy Transfer.

A novel technique, two-dimensional (2D) polarization single-molecule imaging, is presented. It is based on measurements and analysis of fluorescence intensity as a function of excitation and emission polarization angles. The technique allows recording of full information on the steady-state polarization properties of fluorescent objects. It is particularly suitable for application to single multichromophoric systems (molecules or nanoparticles) with energy transfer (ET) between different chromophores (e.g., single fluorescent pi-conjugated polymer chains). The 2D polarization data simultaneously provide information on the conformation of the system and the efficiency of its internal excitation ET. The technique is used to characterize single chains and different kinds of chain aggregates of different conjugated polymers at different temperatures. The 2D polarization measurements reveal a dramatic difference in ET taking place in these systems. Clear temperature dependence of ET is observed for individual aggregates as well as for their statistical ensembles. Also, a dependence on solvent and aggregate size is shown. Additionally, extensive "traditional one-dimensional" polarization results on the polarization anisotropy of fluorescence excitation and emission are presented. These results and findings are discussed in relation to internal organization of the nano-objects under study

Ligand Binding Studied by 2D IR Spectroscopy Using the Azidohomoalanine Label

We explore the capability of the azidohomoalanine (Aha) as a vibrational label for 2D IR spectroscopy to study the binding of the target peptide to the PDZ2 domain. The Aha label responds sensitively to its local environment and its peak extinction coefficient of 350-400 M(-1) cm(-1) is high enough to routinely measure it in the low millimolar concentration regime. The central frequency, inhomogeneous width and spectral diffusion times deduced from the 2D IR line shapes of the Aha label at various positions in the peptide sequence is discussed in relationship to the known X-ray structure of the peptide bound to the PDZ2 domain. The results suggest that the Aha label introduces only a small perturbation to the overall structure of the peptide in the binding pocket. Finally, Aha is a methionine analog that can be incorporated also into larger proteins at essentially any position using protein expression. Altogether, Aha thus fulfills the requirements a versatile label should have for studies of protein structure and dynamics by 2D IR spectroscopy

Hydrophobic Collapse in N-Methylacetamide Water Mixtures

Aqueous N-methylacetamide solutions were investigated by polarization resolved pump probe and 2D infrared spectroscopy (2D IR), using the amide I mode as a reporter. The 2D IR results are compared with molecular dynamics simulations and spectral calculations to gain insight into the molecular structures in the mixture. N-Methylacetamide and water molecules tend to form clusters with "frozen" amide I dynamics. This is driven by a hydrophobic collapse as the methyl groups of the N-methylacetamide molecules cluster in the presence of water. Since the studied system can be considered as a simplified model for the backbone of proteins, the present study forms a convenient basis for understanding the structural and vibrational dynamics in proteins. It is particularly interesting to find out that a hydrophobic collapse as the one driving protein folding is observed in such a simple system

Simulation of vibrational energy transfer in two-dimensional infrared spectroscopy of amide I and amide II modes in solution

Population transfer between vibrational eigenstates is important for many phenomena in chemistry. In solution, this transfer is induced by fluctuations in molecular conformation as well as in the surrounding solvent. We develop a joint electrostatic density functional theory map that allows us to connect the mixing of and thereby the relaxation between the amide I and amide II modes of the peptide building block N-methyl acetamide. This map enables us to extract a fluctuating vibrational Hamiltonian from molecular dynamics trajectories. The linear absorption spectrum, population transfer, and two-dimensional infrared spectra are then obtained from this Hamiltonian by numerical integration of the Schrödinger equation. We show that the amide I/amide II cross peaks in two-dimensional infrared spectra in principle allow one to follow the vibrational population transfer between these two modes. Our simulations of N-methyl acetamide in heavy water predict an efficient relaxation between the two modes with a time scale of 790 fs. This accounts for most of the relaxation of the amide I band in peptides, which has been observed to take place on a time scale of 450 fs in N-methyl acetamide. We therefore conclude that in polypeptides, energy transfer to the amide II mode offers the main relaxation channel for the amide I vibration.

Enhancing signal detection and completely eliminating scattering using quasi-phase-cycling in 2D IR experiments

We demonstrate how quasi-phase-cycling achieved by sub-cycle delay modulation can be used to replace optical chopping in a box-CARS 2D IR experiment in order to enhance the signal size, and, at the same time, completely eliminate any scattering contamination. Two optical devices are described that can be used for this purpose, a wobbling Brewster window and a photoelastic modulator. They are simple to construct, easy to incorporate into any existing 2D IR setup, and have attractive features such as a high optical throughput and a fast modulation frequency needed to phase cycle on a shot-to-shot basis