Herpes simplex virus (HSV) infects skin and genital mucosa and is restricted to epithelial cells and neurons. Immune cells that control the virus include CD8 T cells, and in order to access infected cells they must traffic to the tissue. Previous research has shown that certain chemokines (Ch) and ligands may have an effect in HSV-specific T cell homing. In a study published in Journal of Virology by researchers at the University of Washington and the Vaccine and Infectious Disease Division at Fred Hutch, lead by Dr. Koelle measured Ch expression on HSV- specific CD8 T cells looking for potential correlates for homing to infected tissues.
During viral infection by HSV, lymphocytes traffic to the skin then enter the tissue. A previous paper by this group showed that cutaneous lymphocyte-associated antigen (CLA) is expressed on HSV-specific CD8 T cells and its interaction with the ligand (E-seleectin ligand, ESL) mediates adhesion of the cells to the inflamed vascular endothelium. T-cells leave the circulatory system for the tissue by interactions between Ch and their lymphocyte transmembrane chemokine receptor (ChR). CD8 T cells that leave the circulatory system and enter tissue are called tissue resident memory cells (TRM) and often participate in controlling recurring infection. To better understand this process, the group worked to confirm CLA expression and identify Ch/ChR pairings that are important in recruiting HSV-specific CD8 T cells to the site of infection in human tissue. Flow cytometry was used to measure expression of possible skin homing Ch on circulating cells and to compare the results to control cells, and to CD8 T cells specific for control infections (Epstein-Barr virus, EBV and cytomegalovirus, CMV). To test the transcript levels of these Chs, microarray analysis was used and matched to controls. For this study, 19 different HSV-specific CD8 T cell populations from 16 donors were used and compared to bulk CD8 T cells and EBV/CMV-specific T cells to elucidate differences in the populations.
Looking at CLA expression on CD8 T cells specific for HSV, EBV, CMV, and bulk memory, overexpression of CLA was seen on HSV-specific CD8 T cells compared to the other groups (see figure left panel). This high expression suggests that the CLA helps the CD 8 HSV-specific cells home to the inflamed tissue, which expresses CLA ligand, during viral infection. To identify ChR on HSV-specific CD8 T cells, samples were screened by flow cytometry for HSV-tetramer and anti-ChR double positive cells. Comparing control groups to HSV-specific CD8 T cells found higher expression of CXCR 3 compared to all controls, higher CCR10 compared to the other viruses and higher expression of CCR4 compared to CMV. If this data is taken together with CLA expression on the HSV-specific CD8 T cells, a correlation between CLA positive and CCR10 high cells was seen. However, in HSV-specific CD8 T cells, the expression of CXCR3 was similar in high or low CLA expressers (right panel of figure). Using transcript levels, Ch ligand expression was measured comparing infected samples to healthy control tissue. This allowed for identification of possible ligand/ receptor interactions and thus homing of the HSV-specific CD8 T cells. The mRNA levels for the interferon induced ligands for CXCR3 were increased in HSV infected tissue while the CCR10 ligand levels were equivalent between infected and control samples. This suggests a model where the CCR10 ligand drives initial recruitment of HSV-specific CD8 T cells and that the interferon induced CXCR3 ligands recruit more cells after viral inflammation.
This work has lead to more questions which Dr. Koelle plans to tackle in the future, “We want to know how this stable homing program is mechanistically set within these cells. We suspect there are specific patterns of transcription factor expression and epigenetic changes in the enzymes involved in synthesizing cell-surface CLA, which is actually a complex carbohydrate moiety on the lymphocyte surface created by the concerted action of several enzymes and transporters. Our planned research involves sorting pure CLA-high and CLA-negative cells and studying their transcriptional and epigenetic properties”. The long term goal of this work is to better understand tissue-homing properties and use this knowledge towards vaccine design. In regards to future vaccine properties Dr. Koelle states, “If we could program cells to first home to skin and than to be retained there as a local memory cells, we could provide pre-positioned host defense sentinel cells at sites of microbial infection. By understanding the natural control of skin homing at high resolution in the context of an infection we hope to be able to control this process in the future”.
Funding is provided by the National Institutes of Health and the University of Washington Center for AIDS research (CFAR).
Hensel MT, Peng T, Cheng A, De Rosa SC, Wald A, Laing KJ, Jing L, Dong L, Magaret AS, Koelle DM. 2017. Selective expression of CCR10 and CXCR3 by circulating human HSV-specific CD8 T-cells. J Virol.