The relatively accessible area of genital tissue for observation and sampling have presented an opportunity and a platform to understand the mechanism of peripheral immunity required for effective protection and control of chronic viral infections in humans in vivo. Our close collaboration with Drs. Anna Wald and Christine Johnston at the UW Virology Research Clinic grants access to tissue biopsies from HSV-2 patients. We study these biopsies from different genital regions and at different stages of disease progression. Our immunofluorescence studies can detect HSV-2 virions, lymphocytes and neurons, to name a few, localized at the site of infection in the genital skin and mucosa.
One major innovation of the lab is the development of the rapid cell specific laser capture microdissection (LCM) procedure to quickly identify and precisely isolate single cells (e.g., CD8+ T cells) in tissue according to their anatomic localization. Antigen-specific immuno-LCM results in high quality genetic material and a pure population of cells of interest based on their immunophenotype. As a result, the integrity of cellular RNA from captured cells is high and suitable for downstream functional genomic analyses. We are able to generate gene expression profiles, characterize TCR Vβ clonality and do routine PCR validations with just one round of unbiased whole transcriptome amplification from 50 to 100 captured CD8+ T cells. As there is a unique anatomical localization of HSV-specific CD8+ T cells in the skin and mucosa, we are able to evaluate the specificity of HSV-specific T-cell responses, especially of cells located at the dermal epidermal junction (DEJ) contiguous to the sensory nerve endings in the genitalia, without in vitro manipulation.
Our studies were the first to show that HSV-2 specific tissue resident memory (TRM) T cells persist in genital mucosa for extended time periods, that a subset of these cells resides at the DEJ contiguous to the neuronal endings of sensory nerves, and that these cells are activated and making antiviral cytokines and chemokines during clinically quiescent time periods. These studies also identified a unique phenotype called CD8αα T cells as the TRM cells at the DEJ. Studies are underway to define the natural history of antigenic diversity and functional characteristics of such cells over time.
We have established a technology to combine emulsion-based microfluidics with DNA, RNA and/or protein barcoding that allows recovery of natively paired TCRs (and BCRs) at the repertoire scale. This process is high-throughput, allowing up to 1 million cells per hour to be evaluated in individual 65 picoliter emulsion drops. Cells within droplets are lysed and target mRNA is reverse transcribed with target specific primers in a 2-step process that attaches both molecule specific and droplet specific barcodes to the cDNA. After subsequent recovery, the DNA is sequenced using the Illumina platform. Dual barcoding allows clustering of sequence reads to both molecules and cells of origin. We can also evaluate if direct in vitro co-culture with HSV-2 before single cell sorting allows selection of HSV-2 specific T-cell clones directly from tissue. We can recover thousands of T-cell clones from PBMC and biopsy tissues. The yield, throughput and consistency of the microfluidic single cell sorting approach, especially for identifying CD8+ T cells, are marked improvements over previously used technologies.
Using laser capture microdissection (LCM), we can isolate individual cells from sequential genital biopsies during times of HSV-2 reactivation as well as clinical quiescence. Sequencing the TCR a and b chains from CD8+ T cells found in lesion and healed biopsies allows us to construct reporter TCRs in vitro to evaluate the antigen specificity of TRM CD8+ T cells. We have identified HSV-2 antigens that patient CD8 T cells recognize locally at the site of infection using an HSV-2 ORF library. These TCR reporter genes will help identify which antigens are biologically relevant targets for future HSV vaccines.
HSV-2 DNA is present in several genital tract regions during viral shedding. To specify spatiotemporal HSV dispersion, we have developed a grid system in which daily swabs are taken from 23 separate sites of a patient’s vulva for up to 30 days. During brief asymptomatic episodes, low HSV-2 quantities are confined to a few regions in which both viral load and location fluctuate over time. During longer, lesional episodes, variable viral quantities are detected throughout the genital tract. Even during times of clinical quiescence, the virus is widely dispersed, with heterogeneous viral loads.
Recent progress in cancer immunotherapy has opened the door to use immunological approaches to treat chronic viral infections. Our lab has initiated several preclinical studies to evaluate CAR T cells for treating HIV-1 infection. This is done in collaboration with investigators at Seattle Children’s Hospital, University of Washington and Oregon Health Sciences University. Adoptively transferred autologous Chimeric Antigen Receptor (CAR) T cells can be engineered to 1) be directed to target conserved HIV/SIV epitopes, 2) function independently of MHC, enabling them to potentially target HIV-infected cells that are not effectively cleared by the host’s CTL, 3) retain cytotoxic activity for prolonged time periods, and 4) be designed to traffic to immunologically privileged sites such as B cell follicles, especially when they express germinal center homing markers such as CXCR5. Engineered CAR T cells offer the unique opportunity to target potent anti-HIV effector cells including anti-HIV-1 CD4+ CAR T cells to sites associated with viral persistence. The lab is designing, developing and delivering several novel engineered T cells to evaluate their ability to improve remission and eventually eradication of lentivirus infection in nonhuman primates (NHP).