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Introduction Lipid metabolism plays a crucial role in both innate and adaptive immune responses. Blood lipids are well known biomarkers of heart disease and atherosclerosis. Given that immune responses are involved in atherosclerosis development and progression, it is of utmost importance to explore the mutual relationship between circulating lipid measures and immunology markers, to better manage cardiovascular disease (CVD). Purpose The aim of this work was to evaluate relationships of distinct immune cell subtypes and circulating lipids in a general population setting. Methods We investigated the associations between 3,734 immune traits and circulating lipids [LDL-C, HDL-C, total cholesterol (TC), triglycerides (TG), non-HDL-C) in 2,056 individuals from the TwinsUK cohort. Immune traits were defined by high-dimensional flow-cytometry which enabled in-depth assessment of all major lymphocyte and myeloid subsets in peripheral blood. Information on immune cell subset frequency (FREQ) and mean fluorescence intensity (MFI) was available. A linear mixed model regression analysis adjusted for family structure, gender, age and BMI was used to examine the associations between immune traits and lipids. We then undertook a sub-analysis in 608 individuals and 131 lipid metabolites quantified using nuclear magnetic resonance spectroscopy to investigate the associations of specific immune traits and lipoprotein particles. Results We observed 18 significant associations (Bonferroni P-value Conclusion The present study provides the first comprehensive picture of lipid-immune cell associations in the circulatory system. We observed a link between expression levels of CD14+ classical monocytes and circulating lipid metabolites. The same lipid metabolites were previously associated with risk of developing myocardial infarction and stroke speculating a link between CD14+ classical monocytes and CVD risk. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): SYSCID - A Systems medicine approach to chronic inflammatory diseases

Summary The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α4β7 integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a β sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the β sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates., Graphical Abstract, Highlights • HIV vaccine candidate protects against SIVmac251 acquisition • V1 deleted envelope immunogens with V2 in α-helical conformation are protective • V2-specific ADCC as correlate of risk • Anti-V1 antibodies interfere with V2-specific ADCC, Immunology; molecular structure; virology

BACKGROUND: Biomarkers of response to immunotherapy are critical to evaluate efficacy and ultimately select patients most likely to benefit from this potentially toxic treatment. Peripheral blood mononuclear cells (PBMC) are easily collected and offer abundant lymphocytes, yet the molecular determinants of cells relevant to a therapeutic response have not been determined. To identify such cells, likely rare, we developed four state-of-the-art flow cytometry assays that quantify 65 unique proteins on single-cells for comprehensive, high-throughput proteomic interrogation of PBMC and tumor-infiltrating lymphocytes. Our goal is to identify biomarkers and predictors of response to immunotherapy as well as to find potential peripheral correlates of immune responses to the tumor itself. METHODS: Molecular determinants of B, NK, DC, classical T, invariant T, NKT, and MAIT immune cell lineages and differentiation stages, as well as functional markers including cytokines, cytokine receptors, activation/senescence markers, proliferation, and markers of cytotoxicity were chosen as potential biomarkers of response. Proteomic analyses were performed on PBMC from healthy donors and patients with GBM who received checkpoint blockade therapy. RESULTS: Changes in cellular composition, cell differentiation and functional status were quantified in this comprehensive analysis of healthy donor and patient PBMC. Cellular expression patterns varied by cell activation and differentiation status; highlighting the importance of detailed subset analyses. Potential biomarkers were selected using linear discriminant analysis to identify proteomic differences between healthy donor and patient, as well as longitudinal patient PBMC samples. CONCLUSIONS: Using cutting-edge flow cytometry, we generated a comprehensive analysis of molecular and cellular changes that occur in PBMC in response to immune checkpoint blockade. Additionally, we identify sources of interpatient variation that may obscure subtle proteomic changes. This technological advance provides high-throughput proteomic analysis of live lymphocytes, which can then be sorted for downstream molecular analyses: a critical step in biomarker validation and discovery.

Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines.

Although cryopreserved cell specimens are used throughout biomedical research, the process for thawing samples is labor-intensive and prone to error. Here we describe a small laboratory device that couples an uncapped vial of frozen cells to a conical tube containing warm cell culture media. The entire complex is loaded directly into a centrifuge; within 5min, cells are thawed and diluted out of toxic cryopreservation medium. The recovery and viability of cells are slightly reduced compared to the common (traditional) method. However, antigen-specific T-cell function is not affected. Since no technician time is required (beyond uncapping of vials), our device allows the parallel processing of as many samples as a centrifuge can hold (up to 96, in some models). Moreover, since the samples are not thawed manually in a water bath, the problems associated with technician-to-technician differences in sample handling are minimized, as is the potential for contamination. Importantly, the elimination of substantial labor involving subjective decisions standardizes this process and can reduce variability in results from cryopreserved specimens.

Measuring virion infectivity is critical for studying and monitoring the process of HIV-1 infection. The easiest and the most common method utilizes reporter cell lines based on the HIV LTR promoter. The early HIV gene product Tat amplifies expression from the LTR; however, there is a background transcriptional activity that is independent of Tat. Furthermore, LTR activity can be influenced by cellular activation states. We have recently constructed a Rev-dependent expression vector, and as a test of this construct’s functionality, we have integrated this vector into a continuous T cell line. This novel indicator cell has no measurable background signal, is not affected by elevated metabolic states, and yet responds robustly to the presence of HIV. The line is able to complete TCID50 assays in 3–5 days, and appears sensitive to both CCR5- and CXCR4-utilizing viruses.

effector memory T-cells (13). CD45RA, CD45RO, and CD127 are differen-tially expressed during T-cell differentiation, while CD25 and PD-1 are expressed inan activation-dependent manner.Finally, CD38 and HLA-DR were included; differential expression of thesetwo T-cell activation markers identifies subsets disparately correlating with diseasecontrol, particularly in HIV-1 infection (14,15).To explore the expression of activation markers on T

Background HIV-responsive expression vectors are all based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the use of LTR-based reporter vectors to cloned cells, where aberrantly high expressing (HIV-negative) cells can be eliminated. Enhancements in specificity would increase opportunities for expression vector use in detection of HIV as well as in experimental gene expression in HIV-infected cells. Results We have constructed an expression vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites that are efficiently used in human cells. It also contains a reading frame that is removed by cellular splicing activity in the absence of HIV Rev. The vector was incorporated into a lentiviral reporter virus, permitting detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of green fluorescence protein (GFP) reporter and by PCR of reporter transcript following HIV infection. The vector displayed full HIV dependency. Conclusion As with the earlier developed Tat-dependent expression vectors, the Rev system described here is an exploitation of an evolved HIV process. The inclusion of Rev-dependency renders the LTR-based expression vector highly dependent on the presence of replicating HIV. The application of this vector as reported here, an HIV-dependent reporter virus, offers a novel alternative approach to existing methods, in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector permits examination of living cells, can express any gene for basic or clinical experimentation, and as a pseudo-typed lentivirus has access to most cell types and tissues.

Because most studies of AIDS pathogenesis in nonhuman primates have been performed in Indian-origin rhesus macaques ( Macaca mulatta ), little is known about lentiviral pathogenicity and control of virus replication following infection of alternative macaque species. Here, we report the consequences of simian-human immunodeficiency virus SHIV-89.6P and SIVmac251 infection in cynomolgus ( Macaca fascicularis ) and rhesus macaques of Chinese origin. Compared to the pathogenicity of the same viruses in Indian rhesus macaques, both cynomolgus and Chinese rhesus macaques showed lower levels of plasma virus. By 9 to 10 months after infection, both viruses became undetectable in plasma more frequently in cynomolgus than in either Chinese or Indian rhesus macaques. Furthermore, after SHIV-89.6P infection, CD4 + T-cell numbers declined less and survival was longer in cynomolgus and Chinese rhesus macaques than in Indian rhesus macaques. This attenuated pathogenicity was associated with gamma interferon ELISPOT responses to Gag and Env that were generated earlier and of higher frequency in cynomolgus than in Indian rhesus macaques. Cynomolgus macaques also developed higher titer neutralizing antibodies against SHIV-89.6 at 10 and 20 weeks postinoculation than Indian rhesus macaques. These studies demonstrate that the pathogenicity of nonhuman primate lentiviruses varies markedly based on the species or geographic origin of the macaques infected and suggest that the cellular immune responses may contribute to the control of pathogenicity in cynomolgus macaques. While cynomolgus and Chinese rhesus macaques provide alternative animal models of lentiviral infection, the lower levels of viremia in cynomolgus macaques limit the usefulness of infection of this species for vaccine trials that utilize viral load as an experimental endpoint.

The development of a human immunodeficiency virus type 1 (HIV-1) vaccine that elicits potent cellular and humoral immune responses recognizing divergent strains of HIV-1 will be critical for combating the global AIDS epidemic. The present studies were initiated to examine the magnitude and breadth of envelope (Env)-specific T-lymphocyte and antibody responses generated by vaccines containing either a single or multiple genetically distant HIV-1 Env immunogens. Rhesus monkeys were immunized with DNA prime-recombinant adenovirus boost vaccines encoding a Gag-Pol-Nef polyprotein in combination with either a single Env or a mixture of clade-A, clade-B, and clade-C Envs. Monkeys receiving the multiclade Env immunization developed robust immune responses to all vaccine antigens and, importantly, a greater breadth of Env recognition than monkeys immunized with vaccines including a single Env immunogen. All groups of vaccinated monkeys demonstrated equivalent immune protection following challenge with the pathogenic simian-human immunodeficiency virus 89.6P. These data suggest that a multicomponent vaccine encoding Env proteins from multiple clades of HIV-1 can generate broad Env-specific T-lymphocyte and antibody responses without antigenic interference. This study demonstrates that it is possible to generate protective immune responses by vaccination with genetically diverse isolates of HIV-1.

Although a consensus has emerged that an HIV vaccine should elicit a cytotoxic T lymphocyte (CTL) response, the characteristics of an effective vaccine-induced T lymphocyte response remain unclear. We explored this issue in the simian human immunodeficiency virus/rhesus monkey model in the course of assessing the relative immunogenicity of vaccine regimens that included a cytokine-augmented plasmid DNA prime and a boost with DNA or recombinant pox vectors. Recombinant vaccinia virus, recombinant modified vaccinia Ankara (MVA), and recombinant fowlpox were comparable in their immunogenicity. Moreover, whereas the magnitude of the peak vaccine-elicited T lymphocyte responses in the recombinant pox virus-boosted monkeys was substantially greater than that seen in the monkeys immunized with plasmid DNA alone, the magnitudes of recombinant pox boosted CTL responses decayed rapidly and were comparable to those of the DNA-alone-vaccinated monkeys by the time of viral challenge. Consistent with these comparable memory T cell responses, the clinical protection seen in all groups of experimentally vaccinated monkeys was similar. This study, therefore, indicates that the steady-state memory, rather than the peak effector vaccine-elicited T lymphocyte responses, may be the critical immune correlate of protection for a CTL-based HIV vaccine.

BACKGROUND Activated CD4 T-cells differentiate into lineages, commonly referred to as Thelper (Th) subtypes such as Th2, with unique phenotypes, cytokine signatures, and functions. The present panel (Table 2) was designed to identify major Th subsets described to date according to disparate chemokine receptor expression patterns. Immunity to intracellular infections and viruses requires CCR4 CCR6 CCR10 CXCR3 Th1 cells that produce interferon-c (IFN-c) (1,2), while CCR4 1 CCR6 CXCR3 Th2 cells produce interleukin-4 (IL-4), IL-5, and IL-13 and mediate the host defense against helminthes (3). Th9 cells are CCR4 2 CCR6 IL-9-producing cells that could be involved in wound healing of pleural mesothelial cells during Mycobacterium tuberculosis infection (4). CCR4 CCR6 CCR10 CXCR3 Th17 cells are crucial for the host defense against extracellular pathogens, and mainly produce IL-17A, IL-22, and granulocyte-macrophage colony-stimulating factor (GMCSF) (2,5). Finally, CCR4 CCR6 CCR10 Th22 cells produce IL-22, IL-26, and IL13 and are thought to be involved in skin immunity (2,6). However, there is plasticity in the system and some of these phenotypes and functional characteristics, previously thought mutually exclusive, can be expressed by the same cell. One such example is Th17Th1 cells that are CCR4 2 CCR6 CXCR3 and capable of producing both IFNc and IL-17 (5). The affinity maturation of plasma cells in the germinal centers of B-cell follicles requires the help from another specialized CD4 T-cell lineage called follicular helper cells, or TFH (7). These cells express CXCR5, which confers homing to follicular dendritic cell networks within the germinal centers. The gating scheme for evaluating all these subsets with the present panel is illustrated in Figure 1. First, live CD4 T-cells are identified (Fig. 1A), before gating on TFH (Fig. 1B) or other Th lineage cells (Fig. 1C). Additional non-lineage defining

Despite recent discoveries of genetic variants associated with autoimmunity and infection, genetic control of the human immune system during homeostasis is poorly understood. We undertook a comprehensive immunophenotyping approach, analyzing 78,000 immune traits in 669 female twins. From the top 151 heritable traits (up to 96% heritable), we used replicated GWAS to obtain 297 SNP associations at 11 genetic loci, explaining up to 36% of the variation of 19 traits. We found multiple associations with canonical traits of all major immune cell subsets and uncovered insights into genetic control for regulatory T cells. This data set also revealed traits associated with loci known to confer autoimmune susceptibility, providing mechanistic hypotheses linking immune traits with the etiology of disease. Our data establish a bioresource that links genetic control elements associated with normal immune traits to common autoimmune and infectious diseases, providing a shortcut to identifying potential mechanisms of immune-related diseases.