Tuesday, November 5, 2013

A Few Short Term Interactions May Have Long Term Implications for Memory Cell Fate Decisions

Most people understand that when people become infected with a pathogen, like a virus or bacterium, their immune system provides a rapid and robust response to clear the pathogen. However, did you know that there are many different types of immune cells? Some of our immune cells act immediately during infection and then die, and some are long lasting and become memory cells. Vaccines mediate protection against pathogens by stimulating the production of immune cells, some of which eventually become memory cells. When we get a vaccine of an attenuated or inactivated pathogen our memory cells “remember” the pathogen, so if it attempts to invade our bodies again the memory cells can tell the rest of the immune system how to respond; this is called immunological memory. Because memory cells confer long-term protection against pathogens, much effort goes in to understanding how a cell becomes destined to be a memory cell. And while much progress has been made in this field, the complicated signals and interactions involved in deeming a cell a memory cell are still unknown (Kaech et al., 2002).

One important type of immune cell is the T cell. There are many types of T cells, and one of these essential T cells is the CD8+ T cell, sometimes called a cytotoxic or killer T cell. These CD8+ T cells effectively “kill” regular cells in the body that have been infected with a pathogen; this helps stop the pathogen from multiplying and infecting more cells nearby. Because CD8+ T cells play such a central role in preventing infection, a subset of activated CD8+ T cells during infection become memory cells, to provide a swift response upon pathogen re-exposure. But how do these activated CD8+ T cells know if they should be a short-term effector cell, or to become a memory cell to stick around for the long term? A group of researchers thinks that they have found a piece to this puzzle. A recent study shows that the memory fate decision may be a function of the type of activation the T cell receives.

CD8+ T cells, hereon referred to as cytotoxic T cells or just T cells, are specific for a certain antigen. An antigen in this context is a peptide, a piece of protein, from a pathogen that the immune system recognizes as foreign, and serves as an activator for the immune response. A cytotoxic T cell is activated when a foreign peptide for which it is specific is presented to it by another immune cell called a dendritic cell (DC) (Fig. 1). The DC encounters the pathogen, takes up the peptide, and presents it to the naïve (inactivated) cytotoxic T cell. There are two phases to this activation process. In the first, the DC and cytotoxic T cell make many serial brief contacts. In the second phase a long-lasting interaction, about 30 minutes, is made between the cytotoxic T cell and DC cell. It was previously thought that both phases were necessary to activate the cytotoxic T cell to begin to divide, and make effector cells to go out and kill cells infected with pathogen (Hugues et al., 2004). However, this study has found that the interactions in phase I and II determine if a cell will become a short-term or memory cytotoxic T cell.

Fig. 1. DC:T Cell Interaction
The authors of this paper used a mouse model to study T and DC cell interactions because mice have a similar immune system to humans. The scientists isolated DC cells in culture, and treated them with either high (100C) or low (1C) amounts of an antigenic peptide. The DC cells that were 100C are considered to have a high concentration of antigenic peptide on their surface to be presented to their cognate cytotoxic T cell, and the 1C are considered low concentration. The scientists then fluorescently labeled the DCs, and injected 1C or 100C DC cell populations in to mice. Then 18 hours later, the researchers injected the mice with fluorescently labeled cytotoxic T cells that had a receptor specific for the antigenic peptide presented by the injected DCs. The scientists then waited a few hours to allow the T cells to travel to the lymph node where the injected DC cells reside. Then, to visualize the interactions between the T and DC cells, the group used a method called multiphoton intravital microscopy. Using this method, scientists can look through a microscope in to a tissue of a living mouse, and watch interactions between cells. The group from this paper used this technique to count the interactions, and length of interactions, between T cells and DC cells in the lymph node. (**If you would like to know more about multiphoton intravital microscopy, a link is provided at the end of this blog post. Note it is slightly graphic.)

Fig. 2. Graphic of MP-IVM

Using multiphoton intravital microscopy (MP-IVM), the scientists compared the number and length of interactions between T cells and DC cells in mice with 100C DCs or 1C DCs. They found that in the mice injected with only 1C DCs, the T cells were able to interact with the DC, and become activated through phase 1. Remember, phase I consists of multiple transient interactions between the T cell and DC cell. However, these T cells that only interacted with 1C DCs were not able to move on to phase two and have a long-term contact with a DC. On the other hand, T cells in mice with 100C DCs went through phase I more rapidly than the 1C DC mice, and then were able to form a longer phase II-type interaction with a DC. The group then asked, what are the consequences of this? Are T cells that are activated by phase II different than T cells activated only by phase I?

To investigate this, the researchers isolated T cells from mice that were activated by 1C DCs and 100C DCs, and examined their numbers and effector function. They found that at 48 hours, both T cell populations proliferated equally, meaning they divided into more cells at similar rates. However at 96 hours, the T cells that were activated by 100C DCs were much more numerous than T cells activated by 1C DCs. Next they wanted to see if their effector functions were different. They looked at secretion of interferon (IFN) and other cytokines that are secreted by T cells during infection as part of the immune response. At 48 hours cytokine production between the 100C-activated and 1C-activated T cell populations was similar. However, at 96 hours, T cells activated by 100C DCs were secreting more cytokines and IFN. This led the group to posit that perhaps T cells activated by DCs with a higher concentration of peptides, 100C DCs in this study, were activated for longer and could perhaps be part of the memory cell population after infection.

To determine if the types of activated T cells were different in terms of gene expression, the group used a technique called a microarray to probe for activated genes. They did a microarray on 100C DC- and 1C DC-activated T cells at 24 hours to determine differences in gene expression. They found that the expression of about 500 genes were different as early as 24 hours for the two groups of activated T cells! The T cells that were activated by 100C DCs, thus going through both phase I and II activation, expressed a number of genes associated with memory cells, whereas the 1C-DC activated T cells did not. This finding led them to believe that T cells that undergo only phase I activation cannot become memory cells, but T cells that are activated by phase I and II can. To verify this finding, they did another experiment in mice.

The scientists injected T cells activated by 1C DCs into some mice, and 100C DCs in another group of mice. After 30 days, they inoculated the mice with the antigenic peptide that the T cells were specific for. The mice that had 1C DC-activated T cells showed a slow and weak response to the pathogen. However, the mice with T cells activated by the 100C DCs exhibited a swift and robust response to the pathogen. This experiment verified that the T cells activated by both phases were able to form memory cells, but T cells activated only by transient phase I interactions were not. So what does this all mean?

This study’s findings can be broken down in to two main parts. First, as mentioned earlier, it was previously thought that T cells could only be activated if they went through phase I and II activation. However, this study showed that T cells can be activated by just phase I interactions with DCs. They showed that low concentration peptide on DCs cause phase I interaction, and high concentration peptide on DCs cause phase I and II interaction. These two populations of activated T cells have similar abilities to fight pathogen at 48 hours, but the T cells activated by 100C DCs are more effective from 96 hours on. Their second major finding was that this activation pattern, either phase I or phase I and II, is a determinant for memory fate decisions. So what does this mean for the future and why is it important?

Since memory cells confer the protection of vaccines, it is important to understand how cells become memory cells. This finding can help scientists to optimize vaccines, by helping them determine the concentration of antigenic peptides that need to be injected for optimal memory cell production. In addition the findings in this paper lead us to ask some more questions that could be addressed in subsequent studies to better understand memory fate decisions. At 24 hours, they found that the two types of activated T cells already had “decided” if they were going to be memory or short-lived T cells. In the future, we could look at time points earlier than 24 hours to see how soon gene expression changes to become short-lived or memory cells in the two T cell populations. Additionally, not all of the T cells activated by high peptide concentration DCs become memory cells. So the next question is how do the T cells that are capable of becoming memory cells, i.e. cells activated by phase I and II decide to become memory cells? The findings of this paper and future studies addressing questions like the ones posed above will help us to gain a better understanding of memory cell fate decisions, and will have implications for developing more effective vaccines to protect us from disease.

Primary Source:
Henrickson, S.E., Perro, M., Loughhead, S.M., Senman, B., Stutte, S., Quigley, M., Alexe, G., Iannacone, M., et al. (2013). Antigen Availability Determines CD8+ T Cell-Dendritic Cell Interaction Kinetics and Memory Fate Decisions. Immunity, 39, 496-507.

Secondary Sources:
Hugues, S., Fetler, L., Bonifaz, L., Helft, J., Amblard, F., & Amigorena, S. (2004). Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. Nat. Immunol., 5, 1235-1242.

Kaech, S.M., Hemby, S., Kersh, E., & Ahmed, R. (2002). Molecular and functional profiling of memory CD8 T cell differentiation. Cell, 111, 837-851.

Picture Sources:
1. Mak, T.W., Saunders, M.E. (2011). Primer to The Immune Response. Amsterdam: Elsevier.

2. Miller, M.J., Wei, S.H., Cahalan, M.D., & Parker, I. (2002). Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. PNAS, 100, 2604-2609.

**Multiphoton Intraveital Microscopy (MP-IVM) Video**

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