NK ALERT! New Subpopulation Linked in Restriction of B-Cell Transformation by EBV

EBV, or Epstein-Barr Virus, is a pretty common and current virus that affects approximately well over ninety percent of the human adult population. The virus belongs to the herpes family meaning after the bodies defenses respond to the initial viral lytic infection, the virus remains latent in B cells till further proliferation. The virus enters our tonsils after initial transmission via the saliva¹.

Usually, in the vast majority of infected individuals, virus induced CD8⁺ T cells are responsible for the main fight against EBV infection. However, Natural Killer (NK) cells play an important role for the initial innate immune response against EBV. This paper, A Distinct Subpopulation of Human NK Cells Restricts B Cell Transformation by EBV, further explores the abilities of a specific subpopulation of NK cells in preventing B cell transformation by EBV. The specific NK subpopulation, known as CD56brightNKG2A+CD94+CD54+CD62L2, was seen by researchers to produce an interestingly high amount of IFN-γ³. IFN-γ is an important cytokine that is essential for innate and adaptive immunity when dealing with viral and intracellular bacterial infections. The importance of IFN-γ in the immune system stems in part from its ability to inhibit viral replication directly, and most importantly for its immunostimulatory and immunomodulatory effects.³ So what does this mean?

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. 

Drug Resistant HSV, Soon not to be a Problem!

HERPES!

Herpes is nothing new to society and has been a constant nuisance for many years. But a recent article published by the Proceedings of the National Academy of Sciences claims that they have engineered a mutant antibody that is able to effectively neutralize HSV and drug resistant HSV…in mice. Granted not the best news in the world but hey it’s a start!

Before that, a little bit more about the Herpes virus. The Herpes virus can be broken up into two types, HSV-1 which normally causes oral sores and HSV-2, which usually affects the genital region.  Herpes is an enveloped, double strand DNA virus, that is particularly good at causing long-term infection, or latent infections. Once it enters the body it usually persists for the remainder of ones lifetime, with the possibility of the infection becoming active from time to time. Studies show that usually a very minimal amount of people, with a functioning immune system, reactivate the virus, than those who are immunocomprised.

Various antiviral drugs have been developed and have been shown to decrease virus reactivity, such as Acyclovir. However, as attributed to many viruses, they become drug resistant. This drug resistance is particularly seen in individuals with compromised immune systems, and a better treatment for them is continually being researched. Thus we arrive at what the researchers did!

Although still in its early trials, researchers have engineered an antibody known as mAb hu2c. The antibody works by negating the mechanism through which HSV is able to spread from cell to cell. This is a huge step in defeating the virus, being that HSV is able to successfully avoid the immune system through the cell-to-cell spread mechanism. A quick summary of this mechanism: most viruses infect target cells by releasing virions that ultimately infect target cells. In order for this to occur the virus has to face the extracellular environment and in doing so triggers an immune response. Cell-to-cell spread is a mechanism in which a virus can infect other surrounding cells without ever having to face the extracellular environment, and thus can essentially be “hidden” from our immune system. HSV’s ability to cause infection from cell to cell is mediated by viral glycoproteins.

Just Sitting Back, Biding Its Time. Waiting for the Moment to Pounce

     Human cytomegalovirus (HCMV) is a type of herpesvirus. HCMV infects the majority of individuals, but a healthy immune system is able to suppress a primary infection. Upon initial infection HCMV inhibits an antiviral response through the up-regulation of myeloid cell leukemia-1 protein (MCL-1). The induction of MCL-1 expression is dependent on the activation of the ERK-MAPK pathway rather than viral gene expression.(1) The ERK-MAPK pathway induces proteins such as mitogen-activated protein kinase and extracellular signal-regulated kinase. After the primary infection is controlled HCMV establishes a latent infection in undifferentiated bone marrow precursor cells and monocytes cells.(2) The activation of the ERK-MAPK pathway impacts long-term latency in progenitor cells by priming them from the initial virus encounter and enabling them for future reactivation.(1) When the individual with the latent infection becomes immunocompromised such as during transplants, however, the latent HCMV can become reactivated and have detrimental effects. During viral latency there is limited viral gene expression and no detectable virion production. One viral protein expressed during latency is latency unique natural antigen (LUNA) encoded by the latency associated transcript UL81-82ast. Previous studies have suggested that LUNA is involved in the regulation of HCMV reactivation by suppressing lytic transcription and without LUNA may not be able to enter into latency in order to be reactivated later.(2,3) Another viral protein present during latency is UL138. The UL133-138 is required for HCMV infection in endothelial cells.(4) The function of UL138 in productive and lytic infection is understood, but not much is known about the function of UL138 in latent infection.

     Using plasma membrane profiling the authors determined which plasma membrane protein levels were affected by the presence of UL138 during HCMV latency. Multidrug resistance-associated protein-1 (MRP1) was the most dramatically down-regulated of the three proteins affected by the presence of UL138. In fact, in the presence of UL138 MRP1 was undetectable in cells, suggesting that it might be getting degraded.  MRP1 is important in multidrug resistance and handling organic anions. It is found in bone marrow progenitor calls and monocytes, as well as mature leukocytes(5). In infected cells, the deletion of UL138 restored the expression of MRP1, indication that UL138 is necessary for MRP1 down-regulation. UL138 is also known to be present during lytic infection in addition to latent infection and high levels of UL138 48 hours after lysis coincides with decreased levels of MRP1. It was determined that MRP1 mRNA levels were not decreased from UL138 expression, so the down-regulation of MRP1 is probably post-transcriptional. MRP1 produces many cytotoxic agents including vincristine. When vincristine was added to HCMV-latent monocytes the number of latent cells decreased as well as the expression of UL138 RNA. This indicates that vincristine caused the death of latently infected cells. In addition to reducing the number of latently infected cells through killing, vincristine also reduced the number of cells in which reactivation of HCMV occurred after differentiation of monocytes to dendritic cells.

     What is not understood is why UL138 targets MRP1 for degradation in latent cells. One possible reason is that the reduction of MRP1 also reduced LTC4 which is a substrate of MRP1 and therefore inhibited migration of infected cells and impaired the activation of an immune response. The decreased expression of MRP1 could also inhibit the differentiation of precursor cells until the environment for reactivation was sufficient, therefore maintaining latent infection.