T Helper cells are integral to the proper function of our immune system. They contribute directly to eradicating infection as well as providing “help” for a range of other lymnphocytes – CD8+ Cytolytic T Lymphocytes (CTL) being a great example. The CD4+ helper lymphocyte family has been subdivided into various subgroups over the last three decades based on the expression of specific extra- of intra-cellular proteins, the production and release of specific cytokines, as well the cytokines responsible for polarization of the different functionalities derived from a common naive T cell precursor. For the purposes of understanding their biologies, we define these subgroups as unique entities, but it is important to remember that there are many shades of grey in these assignments; many subgroups are very similar and in fact can inter-convert under appropriate conditions.
Here we present four key subsets that control a range of lymphoid functions. The Th1 subset assists the cellular immune network that includes the aforementioned CD8+ CTL. Th2 cells enable antibody-producing B-cells and contribute to the eradication of extracellular pathogens. At the other end of the spectrum are Th17 cells, which drive inflammatory responses and the Treg cells that are responsible for preventing immune reaction(s) against our own proteins – dysregulation of which results in autoimmune diseases like Diabetes Mellitus and Systemic Lupus Erythematosus. Importantly, there is a delicate balance that is actively maintained between these two inter-converting subsets, disruption of which is thought to contribute to the onset of autoimmunity and the loss of cancer-directed immune responses. Understanding what drives and maintains the balance of these two subsets, and the lymphocytes collective, is key to redirecting inappropriate responses and facilitating suppressed functions that will help us provide relief to those less fortunate than us.
At ACD, we believe that understanding how individual cellular components function in a complex environment will help us better define how populations of cells interact and communicate with each other, providing potential entry points for manipulating global function(s). Everything must have a beginning, and for us it starts with deciphering lymphokine functionalities within the lymphoid system. As we develop systems to address these functions, we will share the resulting tools with our colleagues by way of contract profiling and research services. Our initial services are targeting the lymphokines – and their receptors – that polarize lymphocyte development and function. We are pleased to announce the release of the first three bioassays for IL-2, IL-3 and IL-4 function. Like our kinase profiling service, each assay relies upon cellular viability afforded by the respective lymphocyte, and the loss of viability when functional signals emanating from the receptor are disrupted. What follows are brief descriptions of how each lymphokine functions, and how select kinase inhibitors impact these functions. Please look for future editions of our “Tools and Views” newsletter for more in-depth exploration of these functions.
IL-2 is absolutely necessary for the proper development and function of Treg cells. Mice in which the IL-2 gene – or receptor – have been ablated lack Treg cells and develop autoimmune disorders. The IL-2 receptor consists of three subunits, IL-2 initially binds the alpha subunit. This association causes a structural perturbation of IL-2 which facilitates subsequent association with the beta and gamma subunits. These latter two subunits bind to JAK1 and JAK3, respectively – which in turn phosphorylate Signal Transducer and Activator of Transcription family members 3 and 5 (simply STAT3 and STAT5). These proteins form homodimers that promote the transcription of numerous downstream target genes.
IL-3 drives the production of myeloid progenitor cells from a population of multipotent hematopoietic stem cells (mHSC). In conjunction with IL-7, it also drives differentiation of mHSC into lymphoid progenitor cells. The myeloid lineage gives rise to numerous cell types, including the dendritic cells and macrophages that are central players in recognizing foreign invaders and processing them for presentation to the lymphocytes that will lead to their eradication. This cytokine signals through a dimeric receptor to activate JAK2 with downstream phosphorylation of STAT5A/B. Despite the well documented contributions of IL-3 to myeloid development, IL-3 deficient mice have apparently normal lymphoid systems, suggesting that there is either redundancy or functional overlap with respect to this critical role.
IL-4 guides the polarized development of TH2 cells, which among numerous functions promote development of B cells and regulate the isotypes of the antibodies they produce. Interestingly, this cytokine exerts different effects on B-cells at distinct stages of the cell cycle, demonstrating that lymphokine signaling is impacted by the dynamic microenvironment within the cell. This cytokine binds a dimeric receptor that consists of a unique alpha chain and a gamma chain that is shared with the IL-2 receptor. As a consequence of the receptor composition, this cytokine signals through the same Janus kinases (JAK1 and JAK3) used by the IL-2 receptor.
As shown above, IL-2 and IL-3 signal through distinct JAK family members. As such, it would be anticipated that they would respond differently to kinase inhibitors, either those targeting the JAK family (including AZD1480, Tofacitinib and Ruxolitinib), or other kinases that contribute to signaling by the two receptor complexes (SRC-family kinases for instance). As shown in this summary of three independent experiments, we indeed observe differences in the inhibitory profiles (the bigger the circle, the greater the inhibition).
One of the benefits of decades of basic research into lymphocyte function is the availability of numerous T cell lines that are IL-2 dependent for their growth. In this summary of three independent experiments, we have evaluated the responses of two such cell lines (D10.G4.1 and HT-2) to kinase inhibitors when the cells are grown in the presence of IL-2. Those of you familiar with our research on kinase function won’t be surprised to learn that we weren’t surprised by these rather surprising findings. Namely, two different cell lines both responding to IL-2 with notably different responses to a limited panel of kinase inhibitors. Now to be fair, the D10 cells included IL-1 while the HT-2 cells did not. We will of course be repeating these experiments under identical conditions, but we suspect that the differences observed will be preserved!
So there it is, the first glimpse of our inaugural foray into lymphoid function – and the new services (cell-based biological profiling of IL-2, -3 and -4 function as tools to investigate the potential impact – directed or otherwise – of biological or chemical entities). Soon we will be bringing assay services for IL-6 online – signaling our entry into the field of immuno-oncology. Stay tuned for more – and please contact us if you want to learn more about these services, or give them a test drive.