Scientists from the University of Massachusetts Medical School, the Massachusetts Institute of Technology, and the Advanced Centre for Treatment, Research, and Education in Cancer in Mumbai, India, have developed modified fluorescing peptides that undergo a 1,000-fold increase when they bind to major histocompatibility complex proteins in vivo.
Many questions remain about the biological mechanisms that regulate how peptides load onto MHCs. The researchers, whose study appears in the April issue of Nature Chemical Biology, investigated the ability of a dendritic cell to bind peptides during different stages of its development. Unexpectedly, they found that immature dendritic cells can efficiently display peptides.
Contributing to the development of these fluorescing peptides were Lawrence Stern, a professor of pathology, biochemistry, and molecular pharmacology at the University of Massachusetts Medical School, and Barbara Imperiali, a professor of chemistry and biology at the Massachusetts Institute of Technology.
Cell-Based Assay News spoke with Stern and Imperiali this week about their research.
What was your motivation for developing these probes?
BI: In my group, we developed fluorescent probes to study changes that may occur in cells under certain biological conditions. We developed the probes as amino acids that might be able to report changes in the environment upon binding, for example, to another macromolecule. As the amino acids get incorporated into the peptide sequences, they can be used to study any biological system of interest, if they are predicted to bind to a macromolecule.
Larry had been my colleague at MIT, so we had the chance to discuss potential applications for these probes.
LS: We had been looking at MHC II proteins for some time. We were able to measure peptide binding activity very well for purified proteins in vitro, but we did not really have a way to look at peptide-binding activity in a cellular context.
When we saw these environmentally sensitive probes, and we knew that our binding site had a big hydrophobic region that would likely change the environment of a probe that actively bound into it, we thought that we could put the probes together with our experimental system and be able to visualize this binding on a cell surface.
What technologies existed previously for assaying MHC II complexes, and how do your probes improve upon them?
LS: We had been able to look at these MHCs in vitro. We can look at them on a cell using a bioassay readout using a T cell that will get activated by MHC formation. It is very difficult, however, to visualize MHC formation itself.
The advantage here is that we only see the change in fluorescence right when the peptide binds into the protein. It has given us an increase in specificity that we did not have with prior assays.
BI: From the chemical side, there are other environmentally sensitive fluorescent probes, but they do not show the dramatic changes like the probes that we used in our study.
What about plans to further refine or develop these probes for use as a high-throughput screening tool?
BI: At the moment, we are really using them to do basic science. Other people can sort of take the lead when they see the fluorescent signal changes to determine if the probes are useful in their high-throughput runs.
We’re really working on the level of trying to develop the chemistry, but also trying to determine how the probes can be used in a biological system to provide new insights into its function. The important thing is to demonstrate that these probes are useful for binding to cells in real time, without a lot of manipulation. That puts you in the right position to do a cell-based screen.
LS: There is certainly an interest from the biological side in manipulating this process with an eye towards blocking autoimmune processes, or possibly stimulating immune activity to provoke immunity, for example, in a vaccine context.
At the moment we consider this a tool to help us understand the basic biology of the cells. Certainly there is potential for trying to identify this process, which might be useful in immunotherapeutics.
Although we are not planning to conduct such screens, that is certainly a long-term goal in developing this technology.
What commercial partners are you working with?
SI: I’ve worked on and off with some companies in terms of developing the chemistry that we’ve used. The important thing now is that someone makes the amino acids commercially available over the next few months, so that other people can use it.
The truth is that anyone with chemical capabilities can make these probes. All the details are in our paper. Anyone can make the probes with the details we’ve provided.