to BioTechniques free email alert service to receive content updates.
Genital Herpes under Surveillance

05/09/2013
Jim Kling

A covert team of T cells closely watches the movements of the virus that causes genital herpes from an often overlooked location. Where? Find out...


A subtype of CD8αα+ T cells that keep tabs on the virus that causes genital herpes has been found in a location often overlooked by researchers: just under the outer layer of skin.

CD8aa+ (red) resident T cells persist in the dermis in HSV-2 infected areas for over eight weeks after healing. Source: Nature




CD8+ T cells have previously been found at sites of herpes flare-ups long after the lesions had healed, but now researchers have revealed the responsible subtype – known as CD8αα+ -- which respond to herpes simpld virus 2 (HSV-2) outbreaks following the initial infection. Using laser capture microdissection, they isolated these cells from skin biopsies of herpes in order to analyze their function.

These so-called resident T cells have only recently garnered interest from researchers. Previously, immunologists have focused their studies on circulating T cells as the primary agents in immune responses. But in places like the skin, resident T cells are likely to be the first responders to pathogen attacks.

“If people focus only on the blood, they may miss the major players that really do the work,” said Jia Zhu, a research assistant professor in laboratory medicine at the University of Washington and author of a paper published in Nature this week that describes the function of these cells (1).

Resident cells may help to explain why viral reactivation of genital herpes is typically asymptomatic. In addition, the characterization of these cells bolsters hope for a vaccine against herpes simplex virus 2 (HSV-2) as well as other viruses.

In 2007, Larry Corey, the director of the Fred Hutchinson Cancer Research Center, and his team—which includes Zhu—first described these resident T cells (2). Since then, the group has been fine-tuning a cell-type-specific laser capture microdissection technique to further analyze these immune cells.

Laser capture microdissection was originally developed to study cancer cells. But applying the technique to immune cells was a challenge because they are scarcer and more difficult to locate in tissue samples. The signal amplification and analysis steps were particularly difficult. “You have to be really careful,” said Zhu.

The technique doesn’t rely on in vitro methods—such as chemical digestionthat can lead to the loss of many of the cells—a key issue when the number of target cells in the sample is small. In addition, it allows researchers to bypass animal models and work directly with human patients.

To begin with, the group sliced the biopsies into thin samples and stained the CD8+ T cells. They then isolated those cells with their laser microdissection technique. Finally, Corey and colleagues used transcription profiling and T cell receptor complex β genotyping to find clues as to what the different subtypes of CD8+ T cells were doing.

With this information in hand, they next looked for specific T cell subtypes in biopsies of both herpes outbreaks and normal tissue. What they found was that CD8αα+ T cells persist in lesions long after they have healed.

In asymptomatic lesions, where analysis shows viral DNA but no clinical symptoms, multi-color confocal microscopy revealed two to four CD8αα+ T cells clustered around a single virally-infected keratinocyte. In active lesions, however, the tables were turned; a single CD8αα+ T cell was surrounded by tens to hundreds of infected cells. “That’s why those resident cells are important, because they provide immediate protection and are right on the spot [of an outbreak],” said Zhu.

Now, Corey and colleagues are developing a version of their laser microdissection technique that can isolate a single cell. “You don’t need a lot of material. If our single cell [version] works, you’ll need even less material, but you’ll still be able to study in vivo function [of immune cells],” said Zhu.

References

1. Zhu, J., T. Peng, C. Johnston, K. Phasouk, A. S. Kask, A. Klock, L. Jin, K. Diem, D. M. Koelle, A. Wald, H. Robins, and L. Corey. 2013. Immune surveillance by CD8αα + skin-resident T cells in human herpes virus infection. Nature advance online publication (May).

2. Zhu, J., D. M. Koelle, J. Cao, J. Vazquez, M. L. Huang, F. Hladik, A. Wald, and L. Corey. 2007. Virus-specific CD8+ t cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation. The Journal of Experimental Medicine 204(3):595-603.

Keywords:  cell biology immunology