Selective retention of virus-specific tissue-resident T cells in healed skin after recovery from herpes zoster

Herpes zoster is a localized skin infection caused by reactivation of latent varicella-zoster virus. Tissue-resident T cells likely control skin infections. Zoster provides a unique opportunity to determine if focal reinfection of human skin boosts local or disseminated antigen-specific tissue-resident T cells. Here, we show virus-specific T cells are retained over one year in serial samples of rash site and contralateral unaffected skin of individuals recovered from zoster. Consistent with zoster resolution, viral DNA is largely undetectable on skin from day 90 and virus-specific B and T cells decline in blood. In skin, there is selective infiltration and long-term persistence of varicella-zoster virus-specific T cells in the rash site relative to the contralateral site. The skin T cell infiltrates express the canonical tissue-resident T cell markers CD69 and CD103. These findings show that zoster promotes spatially-restricted long-term retention of antigen-specific tissue-resident T cells in previously infected skin.


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Sex of study subjects is listed in Supplementary table 1, and stated in aggregate in the Results section (10 were male and 8 were female). We did not include sex-based analyses of our data because: 1) we have a small sample size, which limits these comparisons, and 2) there is no consistent association of herpes zoster with sex {see PMID 27382600}. Subjects with HZ were recruited to University of Washington Virology Research Clinic approximately one month after HZ onset using the electronic health record query AMALGA system to identify participants. Younger individuals are potentially more likely to participate in clinical research, albeit, our case-finding method was unbiased regarding outreach to persons with a diagnosis of shingles in our health care system. Our median subject age of 47 is close the US-recommended age (50) for receiving the recombinant zoster vaccine: This age was set based on clinical trial data and the age of an upswing in HZ risk. During the recruitment interval (2018-2020) for this study, uptake for either shingles preventative vaccination in the US was quite low in eligible persons (recombinant zoster vaccine for people over 50; live attenuated zoster vaccine for people over 60). Therefore, we think it unlikely that our study was biased to enrolling younger persons due to vaccine-related low shingles rates in older people. We note a few participants in our study received shingles vaccination, but we are unable to assess whether this impacted the results. Study subjects were compensated per skin biopsy and for each blood draw. The study was approved by the University of Washington Institutional Review Board. Participants provided written informed consent.
The study was approved by the University of Washington Institutional Review Board.
No sample size calculation was performed. The number of persons with herpes zoster studied was chosen based on the ability of our clinic to enroll suitable persons willing to participate in a year-long research study. Because there were no preliminary data (in our lab or the literature)

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Antibodies
Antibodies used relevant to our assay endpoints available prior to the design our of research, it was not possible to conduct a power calculation in advance of starting the work. Due to the localized nature of herpes zoster, biopsy of clinically uninvolved, contralateral skin was appropriate to serve as control tissue. Therefore, paired statistical analyses could be used, as detailed in the manuscript. Two-sided statistical tests were used to preclude any a priori assumptions about the direction of differences between the two types of biopsies. The significance tests conducted were consistent with large magnitude differences for skin virus-specific T cell responses between the healed zoster and paired control skin tissues. The consistency of differences between zoster and control skin immune tests results (Fig. 3B, Fig. 3C, Fig. 4A, Fig. 4C), for several assays and for both CD4 and CD8 T cells, suggest that the sample size is sufficient.
One study subject was excluded because the subject was adjudicated to not have herpes zoster based on the rash characteristics. Immunology data were not collected from this subject. No other data were excluded from analyses.
Replication was not applicable for several data types reported, including within sample ICS screening and TCR sequence analysis. ICS and TCR sequencing are single-cell assay such that hundreds to hundreds of thousands of individual cells are queried. Anti-VZV antibody ELISA and neutralization assays were performed with 2-fold titration of sera covering 6 concentrations, to improve robustness of measures. IFN-in sera of cultured T cells was measured ELISA assays were performed in duplicate or triplicate within assay, and at least 2 wells needed to exceed defined values to be considered positive. All T cell antigens reported to be reactive at the full-length open reading frame level were observed in within-assay duplicate or triplicate, and at least 2 wells needed to exceed defined values to be considered positive. All T cell epitopes reported were observed to be reactive in at least two separate assays performed on separate days, in addition to being detected in technical replicates within-assay.
There was no randomization since this was not an interventional study and we were studying a time-course following disease symptom resolution in a small study cohort. Covariant analyses are irrelevant to our immunological study.
Blinding was not relevant since was not an interventional study or clinical trial. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.