Wednesday, October 30, 2013

What makes a T cell become a memory T cell?

          CD4+ T cells, also known as helper T cells (Th cells), support B and T cell immune responses (more on helper T cells).  Once activated, Th cells differentiate into different effectors, depending on chemical factors in the local environment, called cytokines.  One of these effectors is Th1, mainly involved in an antiviral response or response combating intracellular pathogens.  After the antigen has been cleared from the system, most of the Th1 cells are eliminated by immune system controls, however, a small number of the Th1 cells survive and differentiate into memory T cells.  Although the process in which Th cells differentiate into Th1 cells has been extensively studied, very little is known about the differentiation of Th1 cells into memory cells!  Knowing the steps involved in memory cell differentiation is important, because not only do they indicate the factors that effect T cell fate, but this knowledge could be used to design better vaccines that are aimed at increasing memory T cell formation!  T cell receptors (TCRs) (more on T cells and TCRs) create signals during activation that impact the differentiation and expansion of the T cell, therefore, there is a possibility that TCR signaling determines the end fate of T cells as well: whether they become long-lived memory T cells or end-stage effectors to be eliminated after the pathogen has been cleared.

            In a recent study, Kim et al investigated the impact of TCR signals on the end fate of Th1 cells.   They determined which TCR-binding characteristics correspond to memory differentiation.  To do this, they cloned TCR sequences obtained in a deep sequencing process, and then they transfected 293 T cells with TCR retroviral expression vectors so they could express these TCRs.  Next, the T cells were infected with GP 66-77 tetramer (an antigen glycoprotein), and the tetramer off rates (the rate at which TCR and pMHC dissociate) and avidity (number of TCR-pMHC interactions occurring) for TCRs were measured.  Some TCRs had high avidity with quick off rates, and some had low avidity with extremely slow off rates.  When compared to TCR survival 8-42 days after infection, the only significant predictor of memory T cell potential was the tetramer off rate.

Monday, October 21, 2013

Immunosupression of Type 1 Diabetes

Physiological changes in T1D vs a healthy person
(inability to control glucose levels due to lack of insulin being produced)

Type 1 diabetes (T1D) is a serious, yet often overlooked, autoimmune disease that affects approximately 3 million Americans.  It is often lumped into the general category of “diabetes” alongside type 2 diabetes, even though T1D is generally more serious and isn’t preventable.  In people with T1D, islet, or beta (β) cells, in the pancreas are slowly destroyed by the body.  These cells are responsible for producing insulin, which controls blood glucose levels and keeps them at a healthy level.  People with T1D have to constantly monitor their blood sugar and inject themselves with insulin in order to control their glucose levels or risk serious complications such as blindness, foot amputation, or even coma.  As of now, there is no cure for the disease.  Recent research has focused on trying to find a way to prevent the body from attacking itself through manipulation of the immune system and/or to restore β cell function.  However, even an improvement in treatment would greatly increase the quality of life for T1D patients.
Creation of Insulin: proinsulin cleaved to
produce active insulin and C-peptide
Autoimmune diseases are more complex to treat since it’s often very difficult to suppress only the part of the immune system that isn’t functioning.   Similar to cancer, it’s hard to kill only the bad cells in the body without affecting the good ones.  Roep et al have taken a step in the direction of successful immunosupression of the bad immune cells involved with T1D.  In order to understand their work, it’s first necessary to understand a little more about insulin and the immune system.  Insulin isn’t synthesized in its functional state.  It is synthesized as pro-insulin and then cleaved into an active portion with the A and B peptides, which is insulin, and an inactive portion, called C-peptide; you can’t have one without the other (as seen in the figure to the left).  When doctors want to measure β-cell function, they look at C-peptide levels.  So the researchers use C-peptide levels to determine if their methods are preventing immune cells from destroying insulin-producing cells.

Wednesday, October 16, 2013

Lack of Endogenous IL-10 Enhances Production of Pro-inflammatory Cytokines and Leads to Brucella abortus Clearance in mice

As we all know cytokines help us fight off pathogens by molecular signaling with the use of IFNs and ILs. IL-10 regulates helps balance pathogen clearance and immune response. Brucella abortus is a chronic inflammatory disease which can be found in humans as well as animals. Previous studies have shown that IL-10 is a critical cytokine for inflammatory response in the host and prevents damage. Another study by Fernandes and Baldwin, showed that anti-IL-10 resulted in up to 10-fold fewer bacteria in the spleen with mice infected with the same strain of Brucella. IL-10 directly affects IL-12 by down regulating it and presents a feedback loop which ensures there is not excessive inflammation. In this current study by Corsetti et. al. published in September 2013 he set out to find out the results of IL-10 in bacterial clearance, inflammatory response, and aftereffects of being infected with Brucella abortus.

In order to test this, two sets of mice were studied, the wild type and the IL-10 KO (knock-out). Blood marrow cells were collected and cultured in DMEM in order to culture bacterial cells. After 10 days, the cells were infected with Brucella abortus and assayed for concentrations of IL-10, IL-12p40, or TNF-alpha. Brucella abortus strain 2308 was grown separately from the laboratory for 3 days and the mice were infected. Five animals from each group were examined at 1,2,3,6, and 14 weeks and spleens were removed. The spleens were plated and colonies were counted for cytokine analysis. To analyze the other factors such as IL-10 and IL-gamma, they stimulated the cells and unstimulated ones were used as negative controls. To test in vivo production of these factors, blood samples were taken and centrifuged, the supernatant was used for cytokine analysis. Real-Time RT-PCR was used on the splenocytes. FACS (Fluorescence activated Cell Sorter) is a type of flow cytometry in which cells in a heterogeneous mixture are sorted out.

A FACS analysis consists of many washes and separation method such as the one presented in the figure. The livers of the mice were also collected at each week period and stained with H and E. Granulomas (inflammation)  were measured using this method.

From this experimentation, we learned that wild-type infected with Brucella abortus presented increased production of IL-10 but none was found in the IL-10 KO mice as expected. IL-10 production was not only found in the dendritic cells but also in the splenocytes showing that it is produced in vivo and in vitro when infected. They determined that there was elevated proinflammatory cytokine production in IL-10 KO dendritic cells. Lack of IL-10 results in increased IL-12 and TNF-alpha production because it usually suppresses it. IL-10 KO cells resulted in less bacteria compared to the WT; by week 14 in the spleen there was no sign of Brucella abortus. All other factors were upregulated in IL-10 KO mice too. IL-10 KO mice has less granuloma in the liver as the infection time increased.

In conclusion, the knockout of IL-10 enhanced the the inflammatory response in Brucella abortus but could have adverse results in a different bacterial infection. The KO IL-10 led to reduction in liver pathology which is regulated by Treg cells and TGF-beta (increased in absence of IL-10). Further studies will be performed on these two key players.

I chose this article because I never thought of a cytokine having a better effect when being knocked out because I thought they all assisted in the immune response. Given this model was performed in mice, it’d be interesting if the same results would come from a human experiment. I believe it’s important to know these effects because it can help in cures of these disease and a key hallmark of this one is inflammation so this may work on other types of infection and even cancers.


Sunday, October 6, 2013

A New Understanding of Memory B Cell Generation in Bacterial Infections

We all know that when we’re sick, our immune system launches a response to help rid our bodies of the invading pathogen. But in addition to the cells generated for immediate pathogen elimination, our immune system also generates a set of cells that stick around for months to years; these cells are called memory cells1. These long-lasting memory cells that are generated during an infection are specific for a particular pathogen, so if that same pathogen tries to invade months later, your memory cells will immediately recognize it. Once the memory cell recognizes that pathogen it can mount a robust immune response, hopefully before you even begin to feel sick. These memory cells are what mediate the protection against pathogens that is generated by vaccination1.
There are two main types of memory cells: memory B cells and memory T cells. Memory T cells are derived from activated T cells during infection, which are responsible for cell-mediated immunity. T cells activate other immune cells upon infection, and kill cells that are infected with a pathogen. Memory B cells are derived from activated B cells, and are important for antibody secretion. Therefore B cells function as a vital part of humoral immunity, or immunity derived form macromolecules in fluid, in this case our bodily fluids. Antibodies bind pathogen to prevent it from entering your own cells, and to signal phagocytic cells (or “eater cells”) to destroy the pathogen by ingestion. When a memory B cell encounters its cognate pathogen upon secondary infection, it divides to form more B cells that begin to secrete antibodies to fight the pathogen.

There are many types of memory B cells, which differ in the type of antibody they produce. When a B cell is fighting an infection, it can undergo something called a class switch, which changes the type of antibodies it secretes. There are five main types of antibodies, aptly name isotypes, and each has a specific function. The IgG memory B cell, which secretes the IgG antibody isotype, has long been thought to be the primary contributor to our memory B cell populations. However in the past few years scientists have found that our memory B cell populations are actually more diverse than originally thought. In one recent study, scientists established an important role for another type of memory B cell generated after bacterial infection, the IgM B cell.

IgM B cells are the first B cells generated during an immune response for a specific pathogen. While some IgM B cells produce antibodies to begin to target the pathogen for destruction, other B cells begin to undergo class switching to produce other types of antibodies, like IgG. These B cells also undergo mutations in the DNA region that codes for the pathogen-binding domain on the antibody so that the pathogen can bind the antibody with a better fit; this is called affinity maturation. Therefore, it has long been thought that memory B cells that have undergone class switching and affinity maturation, like IgG memory B cells, are better suited for response to secondary infection since they bind the pathogen with higher affinity2. But a study published by Yates and colleagues showed that IgM memory B cells, which don’t undergo affinity maturation, are actually a large proportion of our memory B cells generated during a bacterial infection. Furthermore, these IgM cells are required for the generation of IgG responses during secondary antigen challenge.

Caution Females with NMO: NMO-IgG and You!

Our immune system is absolutely extraordinary, and to an extent, even dangerous. Our bodies target antigens, foreign bodies, by activating the immune system’s plasma cells to produce antibodies that can eventually target the antigen. After successful binding to the target antigen, the antibody can trigger additional responses that will further lead to the destruction of the antigen. Though our immune system is our main line of defense, there are times when what aids us, undergoes changes and becomes not only dangerous, but even life threatening. Although a rather common problem that is seen in immunocompromised patients, where our own body’s defenses act against us, this study focused on the effect that Neuromyelitis Optica induced antibodies, NMO-IgG, would have on women of child rearing age.1
Neuromyelitis Optica is an inflammatory, demyelinating disease of the Central Nervous System. The disease causes the presence of NMO-IgG, which is a class of circulating antibodies that researchers found bind to the main water channel protein, aquaporin-4.2 SO WHAT?

Friday, October 4, 2013

T Cells take their shots, but Tumor Cells are too Much to Handle

Many types of cancers exist today in this world. One of the most common types of cancer found in humans is lymphoma. Lymphoma is a type of cancer where the white blood cells called the lymphocytes divide uncontrollably. (3) This form of uncontrollable division leads to what are called tumors. Tumors as most of us know are masses of abnormal cells due to an abnormal growth or division of cells.  The tumor cells are your Villains because they are trying to kill you. In the event of tumor cells attacking, the immune system recruits what are called T cells. The T cells, specifically Cytotoxic T Lymphocytes (CTLs), are the Heroes because they are trying to save you. In a new study, researchers examined the role of CTLs in a type of lymphoma called non-Hodgkin lymphoma, which is more common than the Hodgkin lymphoma. Although knowing the differences is not important in being able to understand the topic of this recent study, the differences between the two lymphomas can be found in the reference of lymphoma. (3) The importance of this paper lies within the interaction of the Cytotoxic T Lymphocytes and the lymphoma tumor cells. The new study recently published in The Journal of Immunology reveals how non-Hodgkin lymphoma tumor cells can actually “stop” the T cells. When we thought things couldn’t get any worse when it comes to cancer. When I mean by “stop”, the amount of tumor cells present determines a threshold at which the CTLs inactivate as well as delete rapidly in an antigen specific manner. Within the study, the researchers try to figure out the causes of how and what inactivates the CTLs.

Before we begin with the paper, here are some quick basics of immunology as well as what the research paper will be about to get a better understanding:

CTLs are called Cytotoxic T Lymphocytes, or CD8 T cells. They are called CD8 because they carry a surface marker protein called CD8 that allows them to recognize a surface protein called MHC class I (a protein expressed by all nucleated cells that presents broadly-specific foreign peptides). For understanding purposes of the T cells, I will be referring the T cells as CTLs just as the researchers call them in the paper. Like I said before, CTLs are a type of white blood cell found in the immune system. They kill cancerous cells, host cells that are infected by viruses, or cells that are damaged in any way. The purpose of having CTLs in your body is so that they monitor your body to destroy the person’s infected/irregular cells by direct cell contact before infected cells can spread throughout your body.

The CTLs kill the tumor cells in a process called apoptosis (same process for killing infected cells) where the tumor cells commit suicide after binding their antigens found on the surface to the TCRs (T Cell Receptors). Depending on the structure of the proteins in the binding sites found between the receptor and antigen, determines the specificity of the interaction. The interaction between an antigen and a receptor is like the binding of a lock and key.

Thursday, October 3, 2013

Why do activated T cells cluster?

Cytotoxic CD8 T cells are highly motile lymphocytes that seek out and destroy specific targets in the immune response.  Upon activation by antigen-binding, T cells aggregate together, forming clusters, but the functional significance of this clustering remains unclear.

Why do T cells aggregate?  CD8 T cell activation involves recognition of an antigen displayed on MHC class I molecules by professional APCs.  Interaction of CD8 T cell with Ag-bound APC is mediated by LFA-1, a molecule expressed on T cells that binds to ICAM-1 on APCs.  The LFA-1/ICAM-1 adhesion facilitates T cell activation by promoting the joining of T cells and APCs, as well as initiating intracellular signaling that enhances TCR-mediated signals to promote T cell proliferation and differentiation.  Because T cells express both LFA-1 and ICAM-1, the interaction between these two receptors also mediates aggregation of activated T cells!  Studies have shown that this aggregation may regulate T cell proliferation and differentiation, but the functional significance of Ag-dependent clustering of T cells remains unknown.

A recent study conducted by Zumwalde et al. (2013) addressed activated T cell aggregation, and was described in Edition 191 of The Journal of Immunology.  In this study, authors used an in vitro APC-independent CD8 T cell stimulation system to investigate the functional significance of aggregate formation during T cell activation.  A line of ICAM-1-deficient mice were bred, and conjugated fluorescent Abs were used for labeling.  Naïve eild-type and ICAM-deficient CD8 T cells were purified by negative selection.  T cells were blocked in vitro from clustering using endotoxin-free anti-LFA-1 Abs.  Flow cytometry and intracellular staining techniques as well as microscopy techniques were used to examine T cells.

Friend or Foe? The Recognition of Microbiota vs. Pathogens

           The basic function of the human immune system is to detect and combat pathogens and harmful foreign bodies that we encounter on a daily basis. Bacteria, viruses, and fungi contain some very different proteins and nucleic acids than humans do, and it is job of the body’s immune cells to recognize when one of those components has penetrated our physical barriers, such as skin and the mucus in our digestive and urogenital tracts. Some things that trigger the immune system are peptidoglycan (a cell wall component of bacteria that is not present in humans), lipopolysaccharide (a bacterial cell membrane component also not present in humans), and double-stranded RNA (which encodes the genetic information of many viruses) (Mak & Saunders, 2010).
            The basic forms of an immune system are known as the innate immune system. Almost all forms of multicellular life exhibit some properties of an innate immune system, including microscopic organisms such as nematodes. Humans also have an innate branch of their immune system. The job of the innate immune system is to recognize the basic, general components of foreign bodies attacking the cell, such as those described previously. It seems simple, right? If it’s a human protein, leave it alone, and if it isn’t, get rid of it. Research within the past few decades, however, has revealed a gaping hole in this basic principle.
            It has been discovered relatively recently that microbes (aka foreign bodies) are absolutely vital to human life. There are thousands of types of microbes in the different tissues of our bodies, especially our gut, that help us with things like digestion and nutrient absorption (Slonczewski & Foster, 2010). It has been estimated that the number of microbial cells in our bodies is higher than the number of human cells! This estimation makes the hole in immune theory very obvious: if there are so many microbial cells colonizing our bodies, how do our immune cells know what to attack and what to leave alone? In their review article, Hiutung Chu and Sarkis K Mazmanian attempt to provide insight into this problem and perhaps fill in a few pieces of this gap in their 2013 paper “Innate Immune Recognition of the Microbiota Promotes Host-Microbical Symbiosis”.
            The basic argument of this article is that part of the innate immune system, a set of receptors called Pattern Recognition Receptors (PRR’s) evolved specifically to facilitate communication between symbiotic organisms and human cells. PRR’s have thus far been categorized as cell membrane receptors that recognize general features of many different pathogens (Pathogen-Associated Molecular Patterns, or PAMPs). Innate immune cells such as dendritic cells, which engulf and break down foreign bodies, express many PRR’s on their cell membranes. The authors suggest that the evolutionary purpose of PRR’s is not primarily to detect pathogenic cell components and spur phagocytosis of these foreign bodies, but to communicate with symbiotic bacteria and prevent activation of the immune response.


Plasmacytoid dendritic cells: Friend or Foe?

Plasmacytoid dendritic cells (look a lot like plasma B cells but express different proteins on their cell surface) are important interferon-producers in the presence of viral RNA or DNA (an interferon is a cytokine, or a signaling protein released in response to a pathogens).  pDCs are important in linking the innate (nonspecific) and adaptive (specific) immune response through increasing the function of natural killer and T cells, respectfully.

Much is still unknown about pDC function, though.  As discussed in Li et al., there is a debate on whether pDCs inhibit HIV/SIV replication or instead promote chronic immune activations and disease.  In other words, what is the role of pDCs in lentiviral (long incubation period) infections?  Using SIV (Simian immunodeficiency viruses)-infected rhesus monkeys, Li et al. tried to determine the systematic distribution of pDCs using a cell counting/sorting mechanism called flow cytometry.  They also looked their cytokine expression and affect on T cell activation upon infection. 

Tuesday, October 1, 2013

Innate Immune System may be more involved in Multiple Sclerosis than Previously Thought

        Among the many types of autoimmune disorders perhaps the most interesting and least understood disorders are those in which the immune system attacks the central nervous system.  Multiple Sclerosis (MS) is an autoimmune disorder in which invading lymphocytes attack the protective insulation of neurons called myelin.  The disease is neurodegenerative and leaves patients wheelchair bound within a few short decades of its clinical presentation.  While scientists have unraveled many of the pathological hallmarks of the disease, its cause and effective treatment remain elusive. 

Lesions in an MS brain
            A new study in the Journal of Immunology published this month investigates the types of white blood cells that are involved in the attack of white matter (myelin) in the brain.  Previous studies have identified a subset of Helper T cells as a major cause of the pro-inflammatory environment which leads to the ultimate degeneration which is characteristic of the disease.  These cells are part of the adaptive immune systems which targets very specific pathogens and in some cases (like in MS) accidentally targets cells or proteins that are part of the body’s normal function.  However, the new article presents data that demonstrates that innate immune cells called neutrophils may be playing a role in the initial stages of the disease.  These innate immune cells are non-specific to any particular protein but instead identify and attack cells with general molecular patterns associated with invading pathogens.  This is the first time it has been shown that neutrophils have been involved in MS.