Contact between two cells is a key step in the transfer of information during biological processes. However, monitoring dynamic cellular interactions in vivo poses many technical challenges. In a paper in Nature, Pasqual et al.1 report the development of a technique that can track interactions between cells that contact each other through receptor–ligand binding.
A key step in the development of an immune response involves contact between an antigen-presenting cell (APC), such as a dendritic cell, and an immune cell called a T cell. On the APC surface, a receptor called the major histocompatibility complex (MHC) displays a protein fragment known as an antigen. If the antigen is recognized by the receptor on the T cell, an immune response is triggered. Such cellular interaction is essential for the success of vaccination, cancer immunotherapy and the elimination of disease.
Pasqual and colleagues now describe a method that can quantify the interactions between APCs and T cells in vivo. The ability to count the frequency and number of interactions is fundamental to analysing many complex networks. For networks as diverse as Facebook, academic citations and molecular interactions in a biochemical pathway, such measurements are the main way of assessing the importance of an interaction. And yet for the immune system, which is key to good health, a simple tool to allow this has been lacking.
One current approach for mapping cellular interactions involves an enzyme-based labelling technique that measures static connections between neuronal cells grown in culture2. The authors describe an advance on this approach, using a form of enzyme-facilitated interaction mapping that is suited to the transient cellular interactions found in the immune system. Their method tracks the enzymatic transfer of a molecular label containing a small amino-acid tag, attached to an easily monitored molecule such as biotin. The molecular label can be transferred from one cell to another only if the cells are close enough together for an interaction to occur between a receptor and a ligand on the surfaces of the interacting cells. The molecular tag can then be detected by standard cell-analysis methods such as microscopy, or quantified in vitro using fluorescence analysis — tools already available to most biological researchers. The authors refer to this method of tracking a molecular ‘kiss’ between cells as LIPSTIC.
As a testing ground for their approach, Pasqual and colleagues choose a key interaction on the surface of immune cells that is highly dynamic and yet not physically involved in antigen presentation: the contact between a CD40L ligand, which is present on T cells, and its binding partner, the CD40 receptor, on APCs. Using mice, the authors engineered a fusion protein containing a sortase enzyme and CD40L, and generated a version of CD40 that contained glycine amino-acid residues at its amino terminus. The sortase was supplied with a labelling tag that became bound to the enzyme. In this system, when CD40 and CD40L interact, the sortase on the T cell attaches the tag to an N-terminal glycine residue on the APC’s CD40 (Fig. 1).
LIPSTIC offers three major advances for the field. First, it allows the level of cell–cell interactions in the immune system to be quantified — the more APCs and T cells that interact, the higher the amount of cell labelling that is detected. Therefore, LIPSTIC provides a direct measure of a key step in the initiation of an adaptive immune response. It improves on current methods that measure this step indirectly, such as monitoring of the levels of inflammatory cytokine proteins or antibody production, which assess only the downstream effects of such interactions.
Second, LIPSTIC might offer the possibility of identifying the types of T cell with which APCs interact. Interactions between APCs and different T-cell types can determine both the nature and magnitude of an immune response. For example, the degree of activation of T cells that express the protein CD8 can provide a way of assessing the effectiveness of cancer immunotherapy3. Improved understanding of the interactions between T cells and APCs might thus allow the development of more-effective cancer immunotherapies and vaccines.
Third, and perhaps most impressively, the necessary tools and instrumentation for LIPSTIC analysis are readily accessible. This approach could therefore be rapidly implemented without the technology-transfer delays that often slow the adoption of a technical innovation.
To test LIPSTIC’s usefulness for providing biological insights into immune-system function, the authors analysed the APC–T-cell interactions. Surprisingly, they found that these cells have two modes of interaction, although it had been thought that interaction occurs only when an antigen-bound MHC is presented to the T-cell receptor. The authors observed interaction between APCs and T cells that did not require an antigen-loaded MHC; the label was transferred onto cells that were not loaded with antigen. This previously unknown interaction would be difficult to observe without a method such as LIPSTIC. Why does it occur, and what purpose does it serve? The answers could have implications for efforts to improve immune responses.
Despite LIPSTIC’s evident potential, many challenges remain that will determine the impact of this technique on the wider field of study of cell–cell interactions. How well will it work if adapted for use in systems other than those tested by Pasqual and colleagues? Another challenge will be to determine the level of nonspecific background labelling and of labelling errors inherent in the LIPSTIC approach. The ability to assess both the accuracy and precision of a labelling method is needed for all good quantitative tools.
One way in which the authors have already started to address the specificity of labelling is by using a sortase that has a low affinity for N-terminal glycines. However, the body is full of compounds that are similar to N-terminal glycines. Sortase, although specific for protein labelling in vitro, has not previously been used as a labelling tool in as reactive or demanding an environment as a whole organism. The potential for sortase to transfer a labelling tag to other biological entities at a low background level should be examined more fully. Nevertheless, the hitherto secret world of interactions between T cells and APCs can now be dissected and studied. LIPSTIC offers a way of quantifying contact, one of the most mysterious, but key, elements of a cellular network.
Nature 553, 414-415 (2018)