Control of herpes simplex virus type 1 latency in human trigeminal ganglia

Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus that infects humans and, after a primary lytic infection, establishes lifelong latency in the sensory neurons of the trigeminal ganglia (TG). HSV 1 latency is accompanied by a chronic immune cell infiltration of the TG, the infiltrate being mainly composed of CD8+ T cells. These T cells are believed to control viral latency, but cellular and viral factors like viral microRNAs are also considered to play a crucial role in the establishment and maintenance of viral latency. In the present work, it was investigated whether the tissue-infiltrating T cells are clonally expanded, which would indicate that these T cells are activated by antigen. By applying complementarity determining region 3 (CDR3) spectratyping and immunohistochemistry, several clonal expansions were identified in the TG-resident T cells. In addition, several T cells were present that seemed to be unspecific bystander T cells. Strikingly, some expanded T cell clones were present in the right and left TG of the same individual. This strongly suggests that similar antigens are present in both TG and that the infiltration of immune cells to the TG is driven by antigen. The morphology of the TG was investigated by immunohistochemistry and in situ hybridiza¬tion. Analysis of the distribution of T cells throughout the TG provided puzzling results: unexpectedly, most neurons surrounded by T cells did not harbour the only known prominent transcript during latency, the latency associated transcript (LAT). Whether these neurons do actually harbour latent virus was addressed by a combination of LAT in situ hybridisation, T cell immunohistochemistry, and single cell analysis of laser microdissected sensory neurons by PCR. This analysis revealed that only LAT+ neurons were harbouring HSV 1 DNA and viral microRNAs. Also, mRNA for a viral gene product was only detected in LAT+ neurons. All analysed LAT– neurons were devoid of viral microRNAs and DNA of HSV 1. DNA of HSV 2 or varicella-zoster virus (VZV) was not detected in any of the excised neurons. Alto¬gether this indicates that in the vast majority of infected human neurons, HSV 1 latency is not directly controlled by T cells, but rather by cellular or viral factors like the miRNAs. Our data suggest that CD8+ T cells only come into action if these mechanisms are overrun

Control of HSV-1 latency in human trigeminal ganglia—current overview

Although recurrent Herpes simplex virus type 1 (HSV-1) infections are quite common in humans, little is known about the exact molecular mechanisms involved in latency and reactivation of the virus from its stronghold, the trigeminal ganglion. After primary infection, HSV-1 establishes latency in sensory neurons, a state that lasts for the life of the host. Reactivation of the virus leads to recurrent disease, ranging from relatively harmless cold sores to ocular herpes. If herpes encephalitis—often a devastating disease—is also caused by reactivation or a new infection, is still a matter of debate. It is widely accepted that CD8+ T cells as well as host cellular factors play a crucial role in maintaining latency. At least in the animal model, IFNγ and Granzyme B secretion of T cells were shown to be important for control of viral latency. Furthermore, the virus itself expresses factors that regulate its own latency-reactivation cycle. In this regard, the latency associated transcript, immediate-early proteins, and viral miRNAs seem to be the key players that control latency and reactivation on the viral side. This review focuses on HSV-1 latency in humans in the light of mechanisms learned from animal model

HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons.

Major human pathologies are caused by nuclear replicative viruses establishing life-long latent infection in their host. During latency the genomes of these viruses are intimately interacting with the cell nucleus environment. A hallmark of herpes simplex virus type 1 (HSV-1) latency establishment is the shutdown of lytic genes expression and the concomitant induction of the latency associated (LAT) transcripts. Although the setting up and the maintenance of the latent genetic program is most likely dependent on a subtle interplay between viral and nuclear factors, this remains uninvestigated. Combining the use of in situ fluorescent-based approaches and high-resolution microscopic analysis, we show that HSV-1 genomes adopt specific nuclear patterns in sensory neurons of latently infected mice (28 days post-inoculation, d.p.i.). Latent HSV-1 genomes display two major patterns, called "Single" and "Multiple", which associate with centromeres, and with promyelocytic leukemia nuclear bodies (PML-NBs) as viral DNA-containing PML-NBs (DCP-NBs). 3D-image reconstruction of DCP-NBs shows that PML forms a shell around viral genomes and associated Daxx and ATRX, two PML partners within PML-NBs. During latency establishment (6 d.p.i.), infected mouse TGs display, at the level of the whole TG and in individual cells, a substantial increase of PML amount consistent with the interferon-mediated antiviral role of PML. "Single" and "Multiple" patterns are reminiscent of low and high-viral genome copy-containing neurons. We show that LAT expression is significantly favored within the "Multiple" pattern, which underlines a heterogeneity of LAT expression dependent on the viral genome copy number, pattern acquisition, and association with nuclear domains. Infection of PML-knockout mice demonstrates that PML/PML-NBs are involved in virus nuclear pattern acquisition, and negatively regulate the expression of the LAT. This study demonstrates that nuclear domains including PML-NBs and centromeres are functionally involved in the control of HSV-1 latency, and represent a key level of host/virus interaction

Prevalence and predictors of fatigue in glioblastoma: a prospective study

Background The main goal of this study was to assess frequency, clinical correlates, and independent predictors of fatigue in a homogeneous cohort of well-defined glioblastoma patients at baseline prior to combined radio-chemotherapy. Methods We prospectively included 65 glioblastoma patients at postsurgical baseline and assessed fatigue, sleepiness, mean bedtimes, mood disturbances, and clinical characteristics such as clinical performance status, presenting symptomatology, details on neurosurgical procedure, and tumor location and diameter as well as pharmacological treatment including antiepileptic drugs, antidepressants, and use of corticosteroids. Data on fatigue and sleepiness were measured with the Fatigue Severity Scale and the Epworth Sleepiness Scale, respectively, and compared with 130 age- and sex-matched healthy controls. Results We observed a significant correlation between fatigue and sleepiness scores in both patients (r = 0.26; P = .04) and controls (r = 0.36; P < .001). Only fatigue appeared to be more common in glioblastoma patients than in healthy controls (48% vs 11%; P < .001) but not the frequency of sleepiness (22% vs 19%; P = .43). Female sex was associated with increased fatigue frequency among glioblastoma patients but not among control participants. Multiple linear regression analyses identified depression, left-sided tumor location, and female sex as strongest associates of baseline fatigue severity. Conclusions Our findings indicate that glioblastoma patients are frequently affected by fatigue at baseline, suggesting that factors other than those related to radio- or chemotherapy have significant impact, particularly depression and tumor localizatio

Clonal expansions of CD8+ T cells in latently HSV-1-infected human trigeminal ganglia

Herpes simplex virus type 1 latency in trigeminal ganglia (TG) is accompanied by a chronic immune cell infiltration. The aim of this study was to analyse the T-cell receptor β-chain repertoire in latently HSV-1 infected human TG. Using complementarity-determining region 3 spectratyping, 74 expanded β-chain sequences were identified in five TG. No clone appeared in more than one subject. Similar clones were present in the right and the left TG of two subjects. This indicates that these T cells are primed in the periphery and recognise the same antigen in the TG of both side

Communication of CD8+ T cells with mononuclear phagocytes in multiple sclerosis

Objective CD8+ T cells are the most prevailing lymphocyte population in inflammatory lesions of patients with multiple sclerosis (MS) but it is not even known whether they are merely passive bystanders or actively communicate with other cells in the brain. To identify their potential interaction partners, we analyzed CD8+ T cells that contained vectorially oriented cytotoxic granules and analyzed the areas to which the granules pointed. Methods We stained cryo‐sections of active MS lesions of an index patient with antibodies to CD8 and perforin, searched for vectorially oriented perforin granules, and isolated target areas opposing the granules and control areas by laser‐microdissection. From both areas, we analyzed cell‐type specific transcripts by next‐generation sequencing. In parallel, we stained samples from the index‐patient and other patients by four‐color immunohistochemistry (IHC). Results We found transcripts of the mononuclear phagocyte (MP) specific markers CD163 and CD11b only in the microdissected target areas but not in control areas. We validated the finding that MPs are communication partners of CD8+ T cells in MS lesions by classical IHC in samples from the index‐patient and other patients with acute and progressive MS and other inflammatory neurological diseases. Interpretation Because CD163 and CD11b are specifically expressed in MPs, our findings suggest that CD8+ T cells communicate with local MPs. Although it is still unclear if these interactions lead to killing of the communication partners by CD8+ T cells, our data underline that CD8+ T cells play an active role in the pathogenesis of MS

The TAM-TB Assay—A Promising TB Immune-Diagnostic Test With a Potential for Treatment Monitoring

Tuberculosis (TB) epidemiology is changing in Western and Central Europe due to the rise in immigration and refugees fleeing high-TB-burden areas of war and devastation. The change in local demography and the lack of sensitive and specific TB diagnostic and monitoring tools, especially for cases of childhood TB, leads to either missed cases or over-treatment of this group. Here we present a promising new diagnostic approach, the T cell activation marker (TAM)-TB assay, and its performance in a case of extra-pulmonary TB occurring in a 16 year old refugee from Afghanistan. This assay is based on the characterization of 3 activation markers (CD38, HLA-DR, and Ki67) and one maturation marker (CD27) on M. tuberculosis-specific CD4 T cells. It was performed at time-points TO (10 days), T1 (1 month), T2 (6 months), and T3 (12 months) post-treatment initiation. All markers were able to detect active tuberculosis (aTB) within this patient at T0 and reverted to a healthy/LTBI phenotype at the end of treatment. Tantalizingly, there was a clear trend toward the healthy/LTBI phenotype for the markers at T1 and T2, indicating a potential role in monitoring anti-TB treatment in the future. This assay may therefore contribute to improved TB diagnostic algorithms and TB treatment monitoring, potentially allowing for individualization of TB treatment duration in the future

Analysis of the Paired TCR α- and β-chains of Single Human T Cells

Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV) and multiple sclerosis (MS). In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8+ T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8+ T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity

The TAM-TB Assay—A Promising TB Immune-Diagnostic Test With a Potential for Treatment Monitoring

Tuberculosis (TB) epidemiology is changing in Western and Central Europe due to the rise in immigration and refugees fleeing high-TB-burden areas of war and devastation. The change in local demography and the lack of sensitive and specific TB diagnostic and monitoring tools, especially for cases of childhood TB, leads to either missed cases or over-treatment of this group. Here we present a promising new diagnostic approach, the T cell activation marker (TAM)-TB assay, and its performance in a case of extra-pulmonary TB occurring in a 16 year old refugee from Afghanistan. This assay is based on the characterization of 3 activation markers (CD38, HLA-DR, and Ki67) and one maturation marker (CD27) on M. tuberculosis-specific CD4 T cells. It was performed at time-points T0 (10 days), T1 (1 month), T2 (6 months), and T3 (12 months) post-treatment initiation. All markers were able to detect active tuberculosis (aTB) within this patient at T0 and reverted to a healthy/LTBI phenotype at the end of treatment. Tantalizingly, there was a clear trend toward the healthy/LTBI phenotype for the markers at T1 and T2, indicating a potential role in monitoring anti-TB treatment in the future. This assay may therefore contribute to improved TB diagnostic algorithms and TB treatment monitoring, potentially allowing for individualization of TB treatment duration in the future

Depletion and activation of mucosal CD4 T cells in HIV infected women with HPV-associated lesions of the cervix uteri

Background: The burden of HPV-associated premalignant and malignant cervical lesions remains high in HIV+ women even under ART treatment. In order to identify possible underlying pathophysiologic mechanisms, we studied activation and HIV co-receptor expression in cervical T-cell populations in relation to HIV, HPV and cervical lesion status. Methods Cervical cytobrush (n = 468: 253 HIV- and 215 HIV+;71% on ART) and blood (in a subset of 39 women) was collected from women in Mbeya, Tanzania. Clinical data on HIV and HPV infection, as well as ART status was collected. T cell populations were characterized using multiparametric flow cytometry-based on their expression of markers for cellular activation (HLA-DR), and memory (CD45RO), as well as HIV co-receptors (CCR5, alpha(4)beta(7)). Results Cervical and blood T cells differed significantly, with higher frequencies of T cells expressing CD45RO, as well as the HIV co-receptors CCR5 and alpha(4)beta(7)in the cervical mucosa. The skewed CD4/CD8 T cell ratio in blood of HIV+ women was mirrored in the cervical mucosa and HPV co-infection was linked to lower levels of mucosal CD4 T cells in HIV+ women (%median: 22 vs 32;p = 0.04). In addition, HIV and HPV infection, and especially HPV-associated cervical lesions were linked to significantly higher frequencies of HLA-DR+ CD4 and CD8 T cells (p-values < 0.05). Interestingly, HPV infection did not significantly alter frequencies of CCR5+ or alpha(4)beta(7)+ CD4 T cells. Conclusion The increased proportion of activated cervical T cells associated with HPV and HIV infection, as well as HPV-associated lesions, together with the HIV-induced depletion of cervical CD4 T cells, may increase the risk for HPV infection, associated premalignant lesions and cancer in HIV+ women. Further, high levels of activated CD4 T cells associated with HPV and HPV-associated lesions could contribute to a higher susceptibility to HIV in HPV infected women