The cell-cell contacts formed between a lymphocyte and antigen-presenting cell (APC) are a prerequisite for activation. This contact, the immunological synapse, facilitates communication between the cells which leads to the lymphocyte's effector abilities, be it targeted killing or secretion of chemical messengers. However, activation isn't simply a 'one touch' mechanism, but rather requires three distinct signals and plenty of time. For CD 4 T cells (helper T cells), the first signal is binding of its T cell receptor (TCR) to a peptide-MHCII complex on an APC, followed by co-stimulatory contacts between other surface molecules, and finally the secretion of cytokine chemical messengers. This dance between the T cell and APC can last as long as six hours (Huppa et al. 2003)! Once activated helper T cells can differentiate into effector cells and memory cells. Effector helper T cells secrete signaling molecules to help orchestrate the actions of other immune cells (think of them as a conductor of a band). Memory helper T cells can also complete similar functions but these cells persist over time so that if the pathogen attacks again the immune response may be quicker. In a recent study (Ueda et al. 2011), this interaction between helper T cells and APCs was analyzed over time. The authors recognized that six hours is a long time for two cells to be engaged, thus there must important intra- and intercellular events occuring during this period. They analyzed and characterized four distinct stages which occur during this six hour dance-a-thon between the helper T cell and APC.
In this study, the authors used two types of APCs, a class of B cells (CH27) and bone marrow derived dendritic cells (BM-DCs). The helper T cells were isolated from a trangenic TCR mouse line. The APCs were designed to present a specific antigen on their surface, moth cytochrome c (MCC). Simply, the two cell types were incubated together and then froze under high pressure. High pressure freezing helps to faithfully preserve the cell contacts which will be analyzed (Osumi et al. 2006). Three specific microscopy methods were utilized, electron microscopy with 3D tomography (EM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). EM simply involves shooting a beam of electrons through a sample at different angles to visualize the 3D structure of larger molecules within a cell. SEM and TEM are similar variants, SEM usually results in a better resolution for molecules on the surface of a cell while TEM is better for internal examination of a cell.
The first time point analyzed (with TEM) was 30 minutes after the cells met and a very interesting morphology appeared. The T cells only made contact with the APCs via pseudopodia, thin membrane projections which actually protruded into the APC. This stage was adequately dubbed the pseudopodial stage, or stage one. As a control, when T cells were incubated with the APCs in the absence of antigen no pseudopodia resulted. The authors concluded that stage one formation must be antigen-dependent. Thus, any interaction which occurred before pseudopodia was said to be of stage zero. One of the most surprising findings was that the pseudopodia deeply penetrated the APC membrane, nearly coming into contact with the nucleus! Due to the lack of microtubules beneath the pseudopodia, the authors concluded that these protrusions must be actin-dependent. Also interesting, the authors used 3D tomography to reveal that the pseudopodia were conical rather than flat; the latter being the more traditional structure. They hypothesized that this was aimed at increasing the surface area of contact so that helper T cells may survey more antigens being presented on the APC.
To elucidate the structures of stage two, 3D tomography was utilized. During this transition, the mitochondria were in close association with the endoplasmic reticulum. The authors postulated that this was a consequence of intracellular calcium signaling which is a necessary event in activation. Additionally, there were a large number of endosomes which were present at the immune synapse. Endosomes are membrane bound compartments in a cell which mean that the exchange of some sort of material is occurring with the APC. Premature microtubule initiation was also occurring at this stage, suggesting the microtubules are forming the railways necessary to traffic the endosomes and other organelles throughout the cell.
At one to two hours post interaction the authors saw another key intracellular change, the centrioles appeared to accumulate at the immune synapse; this characteristic distinguished stage three. Centrioles are organelles responsible for organizing microtubules and promoting their growth, playing a large role in skeletal support of a cell. This stage was mostly analyzed using TEM because it was intracellular. In the image, a small dot appeared at which seemed to be the initiating site of microtubule growth and radiation. Also at this stage, nothing appeared to be in between the centrioles and the plasma membrane but, during the latter part of this stage, the golgi complex was not lagging far behind the centriole.
Stage four(suplemental movie 9) was the last and was analyzed four hours after cell incubation. At this point, the centrioles began migrating towards the center of the cell and the golgi complex began to take precedent. The golgi complex formed a ribbon structure (suplemental movie ten) of stacks of cisternae by the immune synapse and centrioles. The authors mentioned this stage should be expected because the golgi complex is part of the secretory pathway. This pathway facilitates the release of the aforementioned cytokine messengers necessary as the third signal of lymphocyte activation. At the end the authors completed the same analysis with the BM-DCs as APCs and found the results to be similar.
Ueda's group did an outstanding job correctly characterizing these stages of interactions and provides great insight into what may be occurring at what time point. For instance, the fact that pseudopodia are antigen dependent signifies that the first activation signal may be a necessity before pseudopodia form, so approximately 30 minutes. The golgi complex assembles at four hours, suggesting the third signal, cytokines, may be prepared much later. Also, Ueda et al. (2011) teach a valuable scientific lesson, never generalize a phenomenon without studying it in depth. For example, previous work by Kupfer and Dennert (1984) and Stinchcombe et al. (2006) examined the stages of interaction between cytotoxic T cells and APCs. In these works, centrioles were randomly scattered throughout the cytotoxic T cell. However, Ueda's group didn't take this as a phenomenon representative of all T cells and ultimately found that centrioles localized to the immune synapse in 92% of helper T cells! Not only does this work establish different intracellular modes of activation between helper and cytotoxic T cells but it also suggests further research may be necessary to examine such a process. This work is valuable because it puts helper T cell activation in a time frame for reference; it would be interesting to see how the times for the various stages differ when there is a repeated exposure to a previously encountered antigen. Specific research may be conducted to examine the role of Cx43, a gap junction protein in formation of the immune synapse. This protein facilitates the intercellular communication necessary for activation and its accumulation at the immune synapse must occur during one of these four stages. The interactions between helper T cells and APCs is indeed intricate and elegant, requiring the timely progression through a series of pre-meditated moves.
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