To describe recent findings on the effect of HIV/SIV infection on lymph node viral and T-cell dynamics using imaging-based methodologies.Chronic infection, particularly HIV/SIV, alters dramatically the microenvironment, immune cell frequency, distribution, function and tissue organization of secondary lymphoid tissues. These changes are not always reversible. Over the past few years, the implementation of advanced imaging protocols on human lymph node biopsies as well as on longitudinal lymphoid tissues samples from nonhuman primates (NHP) have provided a wealth of information on how local immune responses evolve over time in response to a persisting retroviral pathogen. Most of the information concerns cytotoxic and helper T cells and viral dynamics. In this review, we detail this information focusing on HIV/SIV infection. We also comment on the gaps that imaging technologies have bridged in our understanding and discuss the translational value of these new findings in the light of emerging therapeutic agendas.Novel imaging platforms allow for dissecting the spatiotemporal dynamics of immune interactions further improving our understanding of the interplay between virus and host and providing important information for designing successful preventive and curative strategies.
Vaccine responses vary by geographic location. We have previously described how HIV-associated inflammation leads to fibrosis of secondary lymph nodes (LNs) and T cell depletion. We hypothesized that other infections may cause LN inflammation and fibrosis, in a process similar to that seen in HIV infection, which may lead to T cell depletion and affect vaccine responses. We studied LNs of individuals from Kampala, Uganda, before and after yellow fever vaccination (YFV) and found fibrosis in LNs that was similar to that seen in HIV infection. We found blunted antibody responses to YFV that correlated to the amount of LN fibrosis and loss of T cells, including T follicular helper cells. These data suggest that LN fibrosis is not limited to HIV infection and may be associated with impaired immunologic responses to vaccines. This may have an impact on vaccine development, especially for infectious diseases prevalent in the developing world.
Introduction: Persons with HIV/AIDS have significant morbidity/mortality due to influenza virus and are thus targeted for prevention through immunization. However, despite numerous reports on post-immunization antibody titers, the effects of vaccination on the lymphoid tissue architecture of patients on cART and local virus reactivation have not yet been explored. Methods/Results: Lymph node (LN) -derived cells and tissues from healthy controls and virologically suppressed HIV+ subjects were obtained pre- and post- vaccination and analyzed by polychromatic flow cytometry, multicolour confocal imaging and histocytometry. The effect of influenza vaccination on viral dynamics was determined in sorted LN-CD4 T cell populations by quantifying the de-novo synthesis of early HIV transcripts. Influenza -specific antibody titers were measured by hemagglutination inhibition assay. Tissue structure and follicular organization was notably different between healthy and HIV+ individuals. Flu vaccination differentially affects the lymphoid architecture as well as the frequency of follicular CD4 T cells in these 2 groups, which may explain previous observations of variable B cell responses to immunization in HIV- infected individuals. Furthermore, vaccination exerted an impact on the actively transcribed virus in follicular CD4 T cell populations. Conclusions: Chronic HIV-infection establishes a fingerprint of lymphoid changes that is irreversible by cART. Our study shows that these changes further perturb the antigenic response towards an important immunogen and set the stage for further mechanistic studies that could elucidate ways to enhance immunogenicity in such high-risk populations.
Abstract The orchestrated interaction between follicular helper CD4 T cells (TFH) and germinal center (GC) B cells is crucial for optimal humoral immunity. However, the regulatory mechanisms behind spatial distribution and function of TFH is not well understood. Here, we studied human TFH cells and found that transitioning to a CD57 hi TFH status was associated with distinct positioning in the GC, phenotype, transcriptional signatures, function and downregulation of their T-cell receptor (TCR). Single cell TCR clonotype analysis indicated a unidirectional transition towards the CD57 hi TFH status, which was marked with drastic changes in the nature of immunological synapse formation where peripheral microclusters become dominant. Lack of central supra molecular activation cluster (cSMAC) formation in TFH synapse was associated with enhanced ubiquitination/proteasome activity in these cells. Our data reveal significant aspects of the tissue organization and heterogeneity of follicular adaptive immunity and suggest that CD57 hi TFH cells are endowed with distinctive programming and spatial positioning for optimal GC B cell help. One Sentence Summary human TFH cell heterogeneity
CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.
Purpose of review The persistence of HIV-1-infected cells, despite the introduction of the combinatorial antiretroviral therapy, is a major obstacle to HIV-1 eradication. Understanding the nature of HIV reservoir will lead to novel therapeutic approaches for the functional cure or eradication of the virus. In this review, we will update the recent development in imaging applications toward HIV-1/simian immunodeficiency virus (SIV) viral reservoirs research and highlight some of their limitations. Recent findings CD4 T cells are the primary target of HIV-1/SIV and the predominant site for productive and latent reservoirs. This viral reservoir preferentially resides in lymphoid compartments that are difficult to access, which renders sampling and measurements problematical and a hurdle for understanding HIV-1 pathogenicity. Novel noninvasive technologies are needed to circumvent this and urgently help to find a cure for HIV-1. Recent technological advancements have had a significant impact on the development of imaging methodologies allowing the visualization of relevant biomarkers with high resolution and analytical capacity. Such methodologies have provided insights into our understanding of cellular and molecular interactions in health and disease. Summary Imaging of the HIV-1 reservoir can provide significant insights for the nature (cell types), spatial distribution, and the role of the tissue microenvironment for its in vivo dynamics and potentially lead to novel targets for the virus elimination.
The continuous development of molecular biology and protein engineering technologies enables the expansion of the breadth and complexity of protein therapeutics for in vivo administration. However, the immunogenicity and associated in vivo development of antibodies against therapeutics are a major restriction factor for their usage. The B cell follicular and particularly germinal center areas in secondary lymphoid organs are the anatomical sites where the development of antibody responses against pathogens and immunogens takes place. A growing body of data has revealed the importance of the orchestrated function of highly differentiated adaptive immunity cells, including follicular helper CD4 T cells and germinal center B cells, for the optimal generation of these antibody responses. Understanding the cellular and molecular mechanisms mediating the antibody responses against therapeutics could lead to novel strategies to reduce their immunogenicity and increase their efficacy.
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