T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers.

BACKGROUND: Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ. METHODS: Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis. RESULTS: Collagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected. CONCLUSIONS: The human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

Histopathological and immunological spectrum in response evaluation of talimogene laherparepvec treatment and correlation with durable response in patients with cutaneous melanoma.

Talimogene laherparepvec (T-VEC) is an intralesional oncolytic virotherapy for patients with irresectable stage III-IVM1a cutaneous melanoma. Although this treatment is considered to mainly act through T cell-mediated mechanisms, prominent numbers of plasma cells after T-VEC treatment have been described. The aim was to investigate how often these plasma cells were present, whether they were relevant in the response to treatment, and if these or other histopathological features were associated with durable response to treatment. Histopathological (granulomas, perineural inflammation, etc.) and immunological features [e.g. B cells/plasma cells (CD20/CD138) and T cells (CD3,CD4,CD8)] were scored and correlated with durable tumor response [i.e. complete response (CR) persisting beyond 6 months after treatment]. Plasmacellular infiltrate was examined with next-generation sequencing and immunohistochemistry (IgG, IgM, IgA, and IgD). Plasma cells were present in all T-VEC injected biopsies from 25 patients with melanoma taken at 3-5 months after starting treatment. In patients with a durable response ( n = 12), angiocentric features and granulomas were more frequently identified compared with patients without a (durable) response ( n = 13); 75% versus 29% for angiocentric features ( P = 0.015) and 58% versus 15% for granulomas ( P = 0.041). There was a class switch of IgM to IgG with skewing to certain dominant Ig heavy chain clonotypes. An angiocentric granulomatous pattern in T-VEC injected melanoma lesions was associated with a durable CR (>6 months). Plasma cells are probably a relevant feature in the mechanism of response but were not associated with durable response.

The architecture of the simian varicella virus transcriptome.

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed nanopore direct RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

Alveolar barrier disruption in varicella pneumonia is associated with neutrophil extracellular trap formation.

Primary varicella-zoster virus (VZV) infection in adults is often complicated by severe pneumonia, which is difficult to treat and is associated with high morbidity and mortality. Here, the simian varicella virus (SVV) nonhuman primate (NHP) model was used to investigate the pathogenesis of varicella pneumonia. SVV infection resulted in transient fever, viremia, and robust virus replication in alveolar pneumocytes and bronchus-associated lymphoid tissue. Clearance of infectious virus from lungs coincided with robust innate immune responses, leading to recruitment of inflammatory cells, mainly neutrophils and lymphocytes, and finally severe acute lung injury. SVV infection caused neutrophil activation and formation of neutrophil extracellular traps (NETs) in vitro and in vivo. Notably, NETs were also detected in lung and blood specimens of varicella pneumonia patients. Lung pathology in the SVV NHP model was associated with dysregulated expression of alveolar epithelial cell tight junction proteins (claudin-2, claudin-10, and claudin-18) and alveolar endothelial adherens junction protein VE-cadherin. Importantly, factors released by activated neutrophils, including NETs, were sufficient to reduce claudin-18 and VE-cadherin expression in NHP lung slice cultures. Collectively, the data indicate that alveolar barrier disruption in varicella pneumonia is associated with NET formation.

Simian Varicella Virus Infects Enteric Neurons and α4ß7 Integrin-Expressing Gut-Tropic T-Cells in Nonhuman Primates.

The pathogenesis of enteric zoster, a rare debilitating complication of reactivation of latent varicella-zoster virus (VZV) in the enteric nervous system (ENS), is largely unknown. Infection of monkeys with the closely related Varicellovirus simian varicella virus (SVV) mimics VZV disease in humans. In this study, we determined the applicability of the SVV nonhuman primate model to study Varicellovirus infection of the ENS. We confirmed VZV infection of the gut in latently infected adults and demonstrated that SVV DNA was similarly present in gut of monkeys latently infected with SVV using quantitative real-time PCR. In situ analyses showed that enteric neurons expressed SVV open reading frame (ORF) 63 RNA, but not viral nucleocapsid proteins, suggestive of latent ENS infection. During primary infection, SVV-infected T-cells were detected in gut-draining mesenteric lymph nodes and located in close vicinity to enteric nerves in the gut. Furthermore, flow cytometric analysis of blood from acutely SVV-infected monkeys demonstrated that virus-infected T-cells expressed the gut-homing receptor α4ß7 integrin. Collectively, the data demonstrate that SVV infects ENS neurons during primary infection and supports the role of T-cells in virus dissemination to the gut. Because SVV reactivation can be experimentally induced, the SVV nonhuman primate model holds great potential to study the pathogenesis of enteric zoster.