The Role of CD40–CD154 Interactions in Autoimmunity and the Benefit of Disrupting this Pathway

Autoimmune disease is associated with adaptive immune component dysfunction such as the B cells and T cells. The cells somehow pass through peripheral tolerance and cause a self reaction. Autoimmunity is determined by genetics, environmental, and hormonal effects that contribute to the disease. Many autoimmune diseases have been studied and examined but the biology behind them are not completely understood.

A major pathway is the CD40-CD40 ligand (CD154) because it is needed to activate many adaptive immune cells such as DC's, B cells, and T cells. This interaction has multiple functions and as costimulatory molecules, they are upregulated in several autoimmune diseases for example SLE (systemic lupus ertyhematosus). It was thought that disabling this interaction could be a new avenue of therapy for autoimmunity.

Caption: The image to the left shows the basic interactions between immune cells in the body when trying to get rid of an antigen. The same interactions occur in autoimmune diseases where the body is attacking self antigens. In this picture, we can see the CD40-CD40L (CD154) interaction described above between a dendritic cell presents antigens and a CD4+ T cell which aims to help the immune response by releasing cytokines.

CD40 provides a help signal to dendritic cells to maturation. The duration of the signal assists in determining the function of the dendritic cells which is displayed in this study. There are various other proteins that can trigger dendritic cells to become activated and thus spark an autoimmune response such as heat shock proteins. These can skew a response using cytokines, interleukins, and different factors. In a mouse model, diabetes was induced when CD40 expression on bone marrow was necessary for heat shock protein induced activation. 

This reaction of CD40-CD154 is not only found in dendritic cells but it also impacts T cells by priming them, polarizing T cells to a certain response.

Caption: The basic polarization of T cells is shown in the following image with different cytokines determining the Th cell developed. 

Th (T helper) cells only express CD154 after they have been stimulated by an antigen which allow them to have this interaction of CD40-CD154; unfortunately over expression of CD154 can lead to autoimmune diseases. This not only occurs in T cells but in B cells as well. The CD40-CD154 interaction enhances CD86 expression on B cells in autoimmunity specifically SLE disease which is a hypersensitive reaction. CD86 contributes to the presentation of self-antigens to T cells thus inducing a negative immune response by the body. 

Myeloid Dendritic Cells Enhance HIV Latency in T Cells

Diagram of the HIV virus

The Human Immunodeficiency Virus commonly known as HIV in the last 30 years has become a worldwide epidemic affecting approximately 23 million people with an additional 4 million cases annually.  This virus systematically disrupts and destroys the host immune system and is the direct precursor to Acquired Immunodeficiency Syndrome or AIDS.  Despite years of intense scientific research there is no cure or vaccine for HIV.  The current form of treatment is continual use of anti-retroviral drugs which can help to keep HIV at bay but are expensive and have side effects.  The virus itself invades a number of different immune cells and inserts its DNA into the DNA of the cell causing it to produce more of the HIV virus.  The virus itself is also toxic to subsets of these cells known as CD4+ T cells which die after the virus has replicated within the cell.  The destruction of these CD4+ T cells mediates the inability of the immune system to fight off other pathogens. 

                A major problem in combating HIV is that after the virus infects a cell it sometimes lays dormant in the cells to be replicated later.  In this case the virus is not immediately killing the cell but it in the meantime escapes normal processes of destruction.  This viral latency allows the virus to remain in the immune system undetected and escape destruction by other immune cells or anti-retroviral therapy.  Despite this common occurrence, the mechanisms underlying this process are relatively unknown.  However, a recent paper has partially uncovered how some CD4+ T cells are latently infected with the HIV virus.  A paper published this month by Evans et al. has demonstrated the myeloid dendritic cells are responsible for the latent infection of inactivated CD4+ T cells.  In this study, researchers co-cultured inactive CD4+ T cells with different types of infected dendritic cells to see whether they would cause a latent infection and if so which dendritic cells specifically.  The resting CD4+ cells were given a green fluorescent protein which gave off a green light when the cells were actively infected with the HIV-like virus.  The researchers then separated out the non-glowing cells which were not actively infected and further examined those.  When these CD4+ T cells were stimulated a small percentage of them turned green when they had not before, indicating that they contained the virus previously but it was not active and thus the virus was in a latent state.  This process allowed scientists to culture the CD4+ T cells with other cells or chemicals to see what factors caused the cells to have an active or latent infection.  Given this, it was soon discovered that infected myeloid dendritic cells caused a latent infection but not other types of dendritic cells.  Myeloid DCs are cells which migrate in the body and encounter pathogens.  These pathogens are then engulfed, chopped up by enzymes within the cell, and represented on the surface of the cell so that T cells may encounter the antigen and form an immune response against it.   Intriguingly it was previously thought that chemicals released by DCs or the environment called cytokines might be playing a role in the process, however, when the chemicals from the DC-T cell interaction were removed and cultured with new CD4+ T cells they did not develop a latent infection indicating that the myeloid DCs must have a physical connection to the T cells for this process to occur.  The connection between the two is an immunological synapse which involves numerous molecular adhesion molecules such as LFA-1 or ICAM.  The same study found that when they blocked these adhesion proteins there was a reduced effect of infection latency among CD4+ T cells but it did not eliminate latent infection completely so more than these two adhesion molecules must be involved.  These would be new potential targets for anti-retrovirals or other drugs.

Depiction of HIV spread from a DC to a T cell

Implications of Chronic Alcoholism for HIV Infection

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Alcohol dependence is the most common form of drug abuse in the US, with around 7% of the population meeting criteria for alcoholism (Grant 1994). We all know that the effects of alcohol are wide-ranging, impacting both behavior and physiology; it impairs judgement, motor skill, etc. It has been known to weaken the immune system, and research in the past few years has linked the effects of alcohol to HIV infection. Common sense tells us that alcohol promotes risky behaviors, including those that increase the possibility of HIV infection (sex, drugs, etc.). Recent interest in binge drinking and HIV has produced data showing alcohol in a binge pattern changed the proportion of immune cells after SIV infection in rhesus macaques, a common animal model for HIV, and may even increase the disease course (Molina et al 2006, Poonia et al 2006). But researchers at the Scripps Research Institute have also showed that chronic alcoholism may generate microenvironments in the body that are more vulnerable to HIV infection.

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To accomplish this study, the researchers developed a protocol in which rhesus macaques self-administered alcohol orally, twice a day for 30 days before infection and during infection. To do this, they mixed alcohol and an orange flavored drink in different ratios, and conditioned the animals to drink the solution (replacing water with the alcohol solution for a short duration). The final concentration given to the animals was 6% alcohol, similar to the alcohol content of most beers, and animals drank enough alcohol per day to cause blood alcohol levels that in humans are greater than the legal driving limit and would decrease motor skill. When the animals were conditioned, they were infected with SIVmac251, a strain of SIV that induces high peak and steady viral loads and is also known to infect the CNS (Burdo et al 2005). SIVs, simian immunodeficiency viruses, are retroviruses that infect non-human primates and produces symptoms and changes in physiology that mirror those induced by HIV infection (it is generally believed that HIV originated from SIV crossing the species barrier to humans).

A New Mechanism of Cell Death by HIV

            It’s a disease that is well known all over the world: human immunodeficiency virus, better known as HIV.  It is often talked about in tandem with acquired immunodeficiency syndrome, or AIDS, which develops in HIV patients over time and is the end stage of the disease.  HIV originated in chimpanzees as simian immunodeficiency virus (SIV) and transferred over into humans in the 1800s.  The first cases in the United States were reported in 1981.  Throughout the 1980s, cases increased dramatically, peaking in the early 1990s.  However, a breakthrough in drug treatment for people living with HIV and HIV prevention campaigns helped to bring the number of cases back down.  The drug therapy known as antiretroviral therapy (ART) is still used today as the main way to help people with HIV live normal lives, hopefully preventing/delaying the progression to AIDS.  They target different points in the HIV virus replication cycle to try and slow down its progression through the body.

Figure 1: HIV replication in a cell.  It is able to dump its
contents into the cell, reverse transcribe its RNA,
integrate it into host DNA, and use the host to create new viral copies.

HIV is a retrovirus, a form of RNA virus that can be inserted into the host DNA, and then uses host cells to replicate.  The virus comes with all sorts of proteins that let it do this.  For example, reverse transcriptase allows the viral RNA to be turned into viral DNA.  Integrase allows it to be inserted into the host DNA to then use the host’s own protein making mechanisms to make new viruses and viral proteins.  Figure 1 shows an outline of HIV replication.  It is possible to trace the viral RNA to viral DNA to host DNA and then back out to spread to other cells. 

HIV also specifically infects a certain class of immune cells known as CD4+ T cells.  CD4+ T cells are called this due to the presence of a cell surface receptor called CD4.  These cells can differentiate into all different subtypes of CD4+ T cells called T helper cells, or Th cells, whose name describes their function: they “help” other immune cells mount responses to pathogens.  CD4+ T cells and all of their progeny are crucial for providing immunity to all sorts of infections, pathogens, and the like.  HIV comes into play and infects the CD4+ T cells.  It creates a chain reaction, infecting, spreading, and slowly killing all the CD4+ T cells in the body.  In simple terms: HIV is slowly knocking out an entire branch of the immune system.  Any further immune function that would need a CD4+ cell to work won’t be able to work once the CD4+ T cells are gone.  Figure 2 lays this out in a graph showing CD4+ T cells in blue, viral RNA copies in read, and time in weeks on the x-axis.  It is possible to see that as the RNA copies go up, the CD4+ T cells go way down.  Many of the ART drugs target these proteins that prevent the HIV from infecting cells as easily or spreading once it has infected a cell.  However, it is still unknown how HIV actually kills CD4+ T cells.

Figure 2: Timeline of HIV infection.  As viral RNA increases,
CD4+ cells decrease.  A latent period exists where the
person may not know they are infected
until their cell count reaches a certain point.

Cooper et al demonstrate one potential way HIV could kill CD4+ T cells.  They first infected cells with HIV and stained for a particular viral protein called p24.  They noticed that the CD4+ T cells that were killed didn’t express this viral protein, while cells that weren’t killed did.  Next, they looked at whether these cells that were lacking expression of this viral protein had been infected with the virus before they died.  More T cells were infected with HIV that also encoded for green fluorescent protein (GFP), which fluoresces green.  GFP is often used as an indicator for protein production. The gene for GFP is placed within the HIV genome, so if HIV proteins are being produced, GFP will also be produced.  If the cells are dead, no GFP will be detectable.  This is a commonly used method to visualize and also quantify protein production.  They analyzed the cells for GFP expression and cell viability, as well as viral cDNA.  Non-viable GFP- cells were found to have copies of viral cDNA.  When viable GFP+ cells were watched over time, the researchers saw that many of these cells eventually died (therefore losing their GFP expression) but retained viral cDNA.  These data together suggest that the cells that were killed died after successful HIV gene expression.

Inflammatory Bowel Disease: More Fun Than It Sounds

Inflammatory bowel disease (IBD) is a defined as inflammation of the intestines.  It comes in two forms: 1) Crohn’s disease (CD) which causes lesions in the entire wall of the bowel and 2) ulcerative colitis (UC) which is characterized as inflammation in the mucosal layer of the colon (hope this did not gross anyone out too much…).   Surprisingly though, the symptoms associated with IBD is NOT caused by a particular pathogen, but by the attack of one’s own immune system.  Therefore, IBS is characterized as an autoimmune disease.  Microbial translocation, where microbial products enter systematic circulation due to loss of barrier integrity of the intestines, has also been observed in IBD, as well as other disorders.  Although great strides have been made to the understanding of IBD, the roles of specific immune cells types are still not completely clear.  In order to better understand IBD, Funderburg et al. (2013) studied if the accompanying inflammation is linked to T-cell activation and thus microbial translocation. 

Funderburg et al. found several important results.  First, blood plasma samples were taken in individuals with IBD and soluble inflammatory markers were measured. CRP (a protein found in the blood during inflammation) and IL-6 (pro-inflammatory cytokine) levels both increased in IBD patients, which is not surprising because IBD is characterized by inflammation. 

Inflamed Colonic Tissue

Role of Mucosal Immunity in HSV Infection

Herpes Simplex Virus (HSV) is one a virus that plagues many people as it is one of the most common viruses. It is transmitted by physical contact such as drinking, sexual contact, kissing, etc. There has yet to be a vaccine or cure to HSV which is interesting as it has been around so long and no cure has yet to be found. We have many defenses such as mucosal immunity but somehow this seems to make it through. We learned about many systems that play a role in mucosal immunity but for HSV the most pertinent would be vaginal muscosa. Mucosa has IgA secretory antibodies that can help defend from pathogens as well as other several cytokines.

In the paper, Role of Mucosal Immunity in HSV Infection, by Kuklin et. al. they were examining the purpose of mucosal immunity in HSV infection. Mice were immunized through the nose with 3 different variables: glycoprotein B, glycoprotein D, or VV. The mice were challenged with different concentrations of plaque forming units (pfu), a unit of particles capable of forming plaques. The mice were examined on a scale of 1-5 based on their severity. In order to measure monoclonal antibodies, they used anti-CD8+ and anti-CD4+ mAbs. This was analyszed with flow cytometry analysis (the use of an electronic detection to detect small particles in fluid such as mAb). This experiment lasted for 30 days.

After the initial immunization experiment they wanted to measure T cell depletion in vivo so they injected them with 2mg/mouse DP (DepoProvera). DP is a progesterone shot and it aims to regulate the period cycle of the female mice; similar to the DP shots women take as birth control. They challenged the mice with 5 X 10 ⁶ pfu of HSV. Anti CD8+ and anti CD4+ mAbs were given to the mice on day 2 and day 4. The mice were sacrificed after 10 days of this second experiment and there cells were combined and analyzed using two-color flow cytometry. Using samples of serum and vaginal secretion, they were able to use the same process as before to measure Ab production. 

Viruses achieve latency by immune-suppressing mechanisms

The majority of people will be exposed to a virus in the Herpes family at some point that will remain in their body for the rest of their life.  The Herpes family is an example of viruses that can establish latency, which is when the virus remain dormant within the host cell and are no longer proliferating, but their viral genome is still present and is being replicated along with the host genome.  In their publication, Human Cytomegalovirus Latency-Associated Proteins Elicit Immune-Suppressive IL-10 Producing CD4 T Cells, Mason et al. examined the mechanism of how these viruses establish latency.  The group focused on a member of the Herpes family, Human cytomegalovirus (HCMV).

HCMV infection is typically asymptomatic, unless the infected person has a compromised immune system.  During the initial infection, there is an extensive CD4+ and CD8+ T cell response (general information on T cells), which controls the active virus.  However, despite the initial immune response, the virus is unable to be cleared and it establishes latency within the host.  The virus establishing latency within the host is problematic because the virus is able to become active (lytic) again, and if this occurs at a time when the immune system is compromised, the individual will experience disease-like symptoms.  In order to clear these viruses from our body, it is important to understand how they are evading our immune system during latency. 

There are different proteins expressed at different stages of HCMV infection.  During the lytic (active) phase, one of the major viral proteins that are recognized by the T cells is gB which is considered one of the immediate early (IE) genes.  When IE genes are absent, this indicates that the virus is latent.  A small amount of HCMV viral genes are expressed during latency, namely UL138 and LUNA.  Interestingly, the viral genes expressed during latency are also expressed during lytic infection.  If there is a T cell response against UL138 and LUNA in both lytic and latent infections, why is it that the infection unable to be cleared in latency?

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.

HIV-1 Integration is Reduced in CD4 T Cells from Elite Controllers

In 2010, 34 million people were living in the world with Human Immunodeficiency Virus (HIV) (1). The current reality, however daunting, is that there are no vaccines available against HIV. Indeed, even modern day treatments are unable to stop progression of HIV to acquired immunodeficiency syndrome, or AIDS. In 2010, 1.8 million people died from AIDS, so clearly this is a high priority area of research (1). Scientists are working all over the globe, continually researching and studying HIV in hopes of developing a vaccine. So far the efforts have been met with continual challenges and not much success. However, there is a group of people referred to as elite controllers, who are able to maintain undetectable levels of HIV-1 replication despite being HIV positive. One could understand why they would be of great interest to scientists, as they provide a model to study for an effective immune response against HIV infection.

A recent article studying elite controllers investigated HIV-1 reverse transcription and integration in directly ex vivo­ isolated CD4 T cells (2). They used CD4 T cells from elite controllers, HIV-1-negative volunteers, and untreated HIV-1 progressors. The quantified CD4 T cell count using negative immunomagnetic selection ex vivo. Next, cells were infected with a yellow fluorescence protein (YFP)-encoding vesicular stomatitis virus G protein (VSV-g) pseudo typed HIV-1 virus. These big words may sound confusing, but just know that this process infects cells, causing only a single round of HIV-1 infection, allowing for detailed assessments of individual early HIV-1 replication steps. Understanding these processes could have very important implications for vaccine development.