Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches.

Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at the cellular and molecular levels. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.

Human lymph node immune dynamics as driver of vaccine efficacy: an understudied aspect of immune responses.

INTRODUCTION: During the last century, changes in hygiene, sanitation, and the advent of childhood vaccination have resulted in profound reductions in mortality from infectious diseases. Despite this success, infectious diseases remain an enigmatic public health threat, where effective vaccines for influenza, human immunodeficiency virus (HIV), tuberculosis, and malaria, among others remain elusive. AREA COVERED: In addition to the immune evasion tactics employed by complex pathogens, our understanding of immunopathogenesis and the development of effective vaccines is also complexified by the inherent variability of human immune responses. Lymph nodes (LNs) are the anatomical sites where B cell responses develop. An important, but understudied component of immune response complexity is variation in LN immune dynamics and in particular variation in germinal center follicular helper T cells (Tfh) and B cells which can be impacted by genetic variation, aging, the microbiome and chronic infection. EXPERT OPINION: We describe the contribution of genetic variation, aging, microbiome and chronic infection on LN immune dynamics and associated Tfh responses and offers perspective on how inclusion of LN immune subset and cytoarchitecture analyses, along with peripheral blood biomarkers can supplement systems vaccinology or immunology approaches for the development of vaccines or other interventions to prevent infectious diseases.

Clonotypic architecture of a Gag-specific CD8+ T-cell response in chronic human HIV-2 infection.

The dynamics of T-cell receptor (TCR)selection in chronic HIV-1 infection, and its association with clinical outcome, is well documented for an array of MHC-peptide complexes and disease stages. However, the factors that may contribute to the selection and expansion of CD8+ T-cells in chronic HIV-2 infection, especially at the clonal level remain unclear. To address this question, we undertook a detailed molecular characterization of the clonotypic architecture of an HLA-B*3501 restricted Gag-specific CD8+ T-cell response in donors chronically infected with HIV-2 using a combination of flow cytometry, tetramer-specific CD8+ TCR clonotyping, and in vitro assays. We show that the response to the NY9 epitope is hierarchical and narrow in terms of T-cell receptor-alpha (TCRA) and -beta (TCRB) gene usage yet clonotypically diverse. Furthermore, clonotypic dominance in shared origin CTL clones was associated with a greater magnitude of cytokine production and antigen sensitivity at limiting antigen dilution as well as enhanced cross-reactivity for known HIV-2 variants. Hence, our data suggest that effector mobilization and expansion in human chronic HIV-2 infection may be linked to the qualitative features of specific CD8+ T-cell clonotypes, which could have implications for viral control and disease outcome.

In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization.

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.

Visualization of HIV-1 reservoir: an imaging perspective.

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.

A follicular regulatory Innate Lymphoid Cell population impairs interactions between germinal center Tfh and B cells.

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-ß. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Acquisition of optimal TFH cell function is defined by specific molecular, positional, and TCR dynamic signatures.

The development of follicular helper CD4 T (TFH) cells is a dynamic process resulting in a heterogenous pool of TFH subsets. However, the cellular and molecular determinants of this heterogeneity and the possible mechanistic links between them is not clear. We found that human TFH differentiation is associated with significant changes in phenotypic, chemokine, functional, metabolic and transcriptional profile. Furthermore, this differentiation was associated with distinct positioning to follicular proliferating B cells. Single-cell T cell receptor (TCR) clonotype analysis indicated the transitioning toward PD-1hiCD57hi phenotype. Furthermore, the differentiation of TFH cells was associated with significant reduction in TCR level and drastic changes in immunological synapse formation. TFH synapse lacks a tight cSMAC (central supra molecular activation Cluster) but displays the TCR in peripheral microclusters, which are potentially advantageous in the ability of germinal center (GC) B cells to receive necessary help. Our data reveal significant aspects of human TFH heterogeneity and suggest that the PD-1hiCD57hi TFH cells, in particular, are endowed with distinctive programming and spatial positioning for optimal GC B cell help.

Immunogenicity of Protein Therapeutics: A Lymph Node Perspective.

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.

Correction: Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

[This corrects the article DOI: 10.1371/journal.ppat.1006902.].

The role of follicular helper CD4 T cells in the development of HIV-1 specific broadly neutralizing antibody responses.

The induction of HIV-1-specific antibodies that can neutralize a broad number of isolates is a major goal of HIV-1 vaccination strategies. However, to date no candidate HIV-1 vaccine has successfully elicited broadly neutralizing antibodies of sufficient quality and breadth for protection. In this review, we focus on the role of follicular helper CD4 T-cells (Tfh) in the development of such cross-reactive protective antibodies. We discuss germinal center (GC) formation and the dynamics of Tfh and GC B cells during HIV-1/SIV infection and vaccination. Finally, we consider future directions for the study of Tfh and offer perspective on factors that could be modulated to enhance Tfh function in the context of prophylactic vaccination.

Follicular CD4 T Helper Cells As a Major HIV Reservoir Compartment: A Molecular Perspective.

Effective antiretroviral therapy (ART) has prevented the progression to AIDS and reduced HIV-related morbidities and mortality for the majority of infected individuals. However, a lifelong administration of ART is necessary, placing an inordinate burden on individuals and public health systems. Therefore, discovering therapeutic regimens able to eradicate or functionally cure HIV infection is of great importance. ART interruption leads to viral rebound highlighting the establishment and maintenance of a latent viral reservoir compartment even under long-term treatment. Follicular helper CD4 T cells (TFH) have been reported as a major cell compartment contributing to viral persistence, consequent to their susceptibility to infection and ability to release replication-competent new virions. Here, we discuss the molecular profiles and potential mechanisms that support the role of TFH cells as one of the major HIV reservoirs.

Altered immune cell follicular dynamics in HIV infection following influenza vaccination.

HIV infection changes the lymph node (LN) tissue architecture, potentially impairing the immunologic response to antigenic challenge. The tissue-resident immune cell dynamics in virologically suppressed HIV+ patients on combination antiretroviral therapy (cART) are not clear. We obtained LN biopsies before and 10 to 14 days after trivalent seasonal influenza immunization from healthy controls (HCs) and HIV+ volunteers on cART to investigate CD4+ T follicular helper (Tfh) and B cell dynamics by flow cytometry and quantitative imaging analysis. Prior to vaccination, compared with those in HCs, HIV+ LNs exhibited an altered follicular architecture, but harbored higher numbers of Tfh cells and increased IgG+ follicular memory B cells. Moreover, Tfh cell numbers were dependent upon preservation of the follicular dendritic cell (FDC) network and were predictive of the magnitude of the vaccine-induced IgG responses. Interestingly, postvaccination LN samples in HIV+ participants had significantly (P = 0.0179) reduced Tfh cell numbers compared with prevaccination samples, without evidence for peripheral Tfh (pTfh) cell reduction. We conclude that influenza vaccination alters the cellularity of draining LNs of HIV+ persons in conjunction with development of antigen-specific humoral responses. The underlying mechanism of Tfh cell decline warrants further investigation, as it could bear implications for the rational design of HIV vaccines.

Lymphoid tissue fibrosis is associated with impaired vaccine responses.

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.

Accumulation of follicular CD8+ T cells in pathogenic SIV infection.

LN follicles constitute major reservoir sites for HIV/SIV persistence. Cure strategies could benefit from the characterization of CD8+ T cells able to access and eliminate HIV-infected cells from these areas. In this study, we provide a comprehensive analysis of the phenotype, frequency, localization, and functionality of follicular CD8+ T cells (fCD8+) in SIV-infected nonhuman primates. Although disorganization of follicles was a major factor, significant accumulation of fCD8+ cells during chronic SIV infection was also observed in intact follicles, but only in pathogenic SIV infection. In line with this, tissue inflammatory mediators were strongly associated with the accumulation of fCD8+ cells, pointing to tissue inflammation as a major factor in this process. These fCD8+ cells have cytolytic potential and can be redirected to target and kill HIV-infected cells using bispecific antibodies. Altogether, our data support the use of SIV infection to better understand the dynamics of fCD8+ cells and to develop bispecific antibodies as a strategy for virus eradication.

Treatment with native heterodimeric IL-15 increases cytotoxic lymphocytes and reduces SHIV RNA in lymph nodes.

B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8+ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (TFH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8+ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8+ cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B+ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals. TRIAL REGISTRATION: ClinicalTrials.gov NCT02452268.

Novel advances on tissue immune dynamics in HIV/simian immunodeficiency virus: lessons from imaging studies.

PURPOSE OF REVIEW: To describe recent findings on the effect of HIV/SIV infection on lymph node viral and T-cell dynamics using imaging-based methodologies. RECENT FINDINGS: 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. SUMMARY: 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.

The Lymph Node in HIV Pathogenesis.

Lymph nodes play a central role in the development of adaptive immunity against pathogens and particularly the generation of antigen-specific B cell responses in specialized areas called germinal centers (GCs). Lymph node (LN) pathology was recognized as an important consequence of human immunodeficiency virus (HIV) infection since the beginning of the HIV epidemic. Investigation into the structural and functional alterations induced by HIV and Simian immunodeficiency virus (SIV) has further cemented the central role that lymphoid tissue plays in HIV/SIV pathogenesis. The coexistence of constant local inflammation, altered tissue architecture, and relative exclusion of virus-specific CD8 T cells from the GCs creates a unique environment for the virus evolution and establishment of viral reservoir in specific GC cells, namely T follicular helper CD4 T cells (Tfh). A better understanding of the biology of immune cells in HIV-infected lymph nodes is a prerequisite to attaining the ultimate goal of complete viral eradication.

Lower Baseline Germinal Center Activity and Preserved Th1 Immunity Are Associated With Hepatitis B Vaccine Response in Treated HIV Infection.

BACKGROUND: Why HIV-infected individuals have poor responses to standard dose and schedule hepatitis B virus immunization is not well understood. METHODS: We compared the serologic and cellular immune profiles of treated HIV-infected individuals with similar durations of infection and preserved CD4 counts (> 350 cells/microliter) by hepatitis B vaccine (HBV) response before and after vaccination. RESULTS: Similar levels of immune activation and plasma cytokine profile were found between non-responders and responders. The baseline plasma levels of CXCL-13, a surrogate of germinal center reactivity, were significantly lower in HBV responders compared to HBV non-responders and were a predictor of both vaccine response and titer. Furthermore, response to HBV vaccination was associated with a significantly higher frequency of circulating IgGhigh memory B cells post vaccination and preserved Th1 antigen-specific T-cell responses. CONCLUSIONS: Taken together, our data suggest that preserved Th1 responses are associated with hepatitis B vaccine response in treated HIV infection.

Follicular CD8 T cells accumulate in HIV infection and can kill infected cells in vitro via bispecific antibodies.

Cytolytic CD8 T cells play a crucial role in the control and elimination of virus-infected cells and are a major focus of HIV cure efforts. However, it has been shown that HIV-specific CD8 T cells are infrequently found within germinal centers (GCs), a predominant site of active and latent HIV infection. We demonstrate that HIV infection induces marked changes in the phenotype, frequency, and localization of CD8 T cells within the lymph node (LN). Significantly increased frequencies of CD8 T cells in the B cell follicles and GCs were found in LNs from treated and untreated HIV-infected individuals. This profile was associated with persistent local immune activation but did not appear to be directly related to local viral replication. Follicular CD8 (fCD8) T cells, despite compromised cytokine polyfunctionality, showed good cytolytic potential characterized by high ex vivo expression of granzyme B and perforin. We used an anti-HIV/anti-CD3 bispecific antibody in a redirected killing assay and found that fCD8 T cells had better killing activity than did non-fCD8 T cells. Our results indicate that CD8 T cells with potent cytolytic activity are recruited to GCs during HIV infection and, if appropriately redirected to kill HIV-infected cells, could be an effective component of an HIV cure strategy.

Novel Imaging Methods for Analysis of Tissue Resident Cells in HIV/SIV.

The use of advanced tissue-imaging methodologies has greatly facilitated the study of molecular mechanisms and cellular interactions in humans and animal models of disease. Particularly, in HIV research, there is an ever-increasing demand for a comprehensive analysis of immune cell dynamics at tissue level stemming from the need to advance our understanding of those interactions that regulate the generation of adaptive antigen-specific immune responses. The latter is critical for the development of vaccines to elicit broadly neutralizing antibodies as well as for the discovery of novel targets for immuno-therapies to strengthen the cytolytic arm of the immune system at local level. In this review, we focus on current and emerging imaging technologies, discuss their strengths and limitations, and examine how such technologies can inform the development of new treatments and vaccination strategies. We also present some perspective on the future of the technology development.