Aging is characterized by a loss of bone marrow hematopoietic tissue, systemic chronic inflammation, and higher susceptibility to infectious and noninfectious diseases. We previously reported the ...tightly regulated kinetics and massive daily production of neutrophils during homeostasis in adult rhesus macaques aged 3 to 19 yr (equivalent to approximately 10 to 70 yr of age in humans). In the current study, we observed an earlier release of recently dividing neutrophils from bone marrow and greater in‐group variability of neutrophil kinetics based on in vivo BrdU labeling in a group of older rhesus macaques of 20–26 yr of age. Comparing neutrophil numbers and circulating cytokine levels in rhesus macaques spanning 2 to 26 yr of age, we found a negative correlation between age and blood neutrophil counts and a positive correlation between age and plasma G‐CSF levels. Hierarchic clustering analysis also identified strong associations between G‐CSF with the proinflammatory cytokines, IL‐1β and MIP‐1α. Furthermore, neutrophils from older macaques expressed less myeloperoxidase and comprised higher frequencies of polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSCs) compared to the young adult macaques. In summary, we observed an earlier release from bone marrow and a reduced production of neutrophils despite the increased levels of plasma G‐CSF, especially in the elderly rhesus macaques. This lower neutrophil production capacity associated with increased production of proinflammatory cytokines as well as an earlier release of less mature neutrophils and PMN‐MDSCs may contribute to the chronic inflammation and greater susceptibility to infectious and noninfectious diseases during aging.
Graphical
Dysregulated neutrophil kinetics, increasing G‐CSF levels, and higher frequency of PMN‐MDSCs accompany chronic inflammation during aging in rhesus macaques.
The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of ...maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αβ T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.
Although the Zika virus (ZIKV) epidemic is subsiding, immune responses that are important for controlling acute infection have not been definitively characterized. Nonhuman primate (NHP) models were ...rapidly developed to understand the disease and to test vaccines, and these models have since provided an understanding of the immune responses that correlate with protection during natural infection and vaccination. Here, we infected a small group of male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques with a minimally passaged Brazilian ZIKV isolate and used multicolor flow cytometry and transcriptional profiling to describe early immune patterns following infection. We found evidence of strong innate antiviral responses together with induction of neutralizing antibodies and T cell responses. We also assessed the relative importance of CD8 T cells in controlling infection by carrying out CD8 T cell depletion in an additional two animals of each species. CD8 depletion appeared to dysregulate early antiviral responses and possibly increase viral persistence, but the absence of CD8 T cells ultimately did not impair control of the virus. Together, these data describe immunological trends in two NHP species during acute ZIKV infection, providing an account of early responses that may be important in controlling infection.
Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α
β
with an anti-α
β
monoclonal ...antibody (Rh-α
β
) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α
β
integrin blockade, SHIV
-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α
β
or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α
β
and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α
β
integrin blockade may prolong virologic control by bNAbs in SHIV
-infected macaques.
HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was ...evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.
Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT ...rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.
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•NKTT320 rapidly activates iNKT in vivo, modulating downstream immune function•In vivo NKTT320 treatment modulates pro- and anti-inflammatory genes•NKTT320 treatment results in activation of innate and adaptive immune subsets•NKTT320 has promise as an immunotherapeutic with translational potential
Biological sciences; Immunology; Components of the immune system; Transcriptomics
Advances in immunology, immuno-oncology, drug discovery and vaccine development demand improvements in the capabilities of flow cytometry to allow it to measure more protein markers per cell at ...multiple timepoints. However, the size of panels of fluorophore markers is limited by overlaps in fluorescence-emission spectra, and flow cytometers typically perform cell measurements at one timepoint. Here we describe multi-pass high-dimensional flow cytometry, a method leveraging cellular barcoding via microparticles emitting near-infrared laser light to track and repeatedly measure each cell using more markers and fewer colours. By using live human peripheral blood mononuclear cells, we show that the method enables the time-resolved characterization of the same cells before and after stimulation, their analysis via a 10-marker panel with minimal compensation for spectral spillover and their deep immunophenotyping via a 32-marker panel, where the same cells are analysed in 3 back-to-back cycles with 10-13 markers per cycle, reducing overall spillover and simplifying marker-panel design. Cellular barcoding in flow cytometry extends the utility of the technique for high-dimensional multi-pass single-cell analyses.
Abstract
Current flow cytometry requires samples to be fully processed and stained for all markers of interest before acquisition. However, assay development become increasingly difficult when many ...colors (>10) are used at once due to spectral spillover. Assay complexity is hampered further by markers that require special cell processing and restrict the choice of fluorophores or antibodies.
We have developed a method to dramatically simplify high-marker flow assays by breaking down a complex measurement into multiple, easier measurements. Our method involves optically barcoding individual cells with semiconductor-based laser particles (LPs) that emit laser light in the infrared. Using LP barcodes and a custom flow cytometer, we track millions of cells across repeated measurements or passes. Cells are de-stained and re-stained after each pass.
We demonstrate several applications of multi-pass flow cytometry. First, we show deep immunophenotyping of human PBMCs, measuring 33 markers with 13 colors over 3 cycles. We validate our method against state-of-the-art, reproducing expected cell types and showing improved data quality due to significantly reduced spillover. Second, we show deep characterization of stimulated mouse T cells, in which an unprecedented 32 markers including activation markers, intranuclear proteins, and intracellular cytokines are detected on the same cell type. Finally, we measure cells before and after harsh cell processing steps to recover signals that would otherwise be lost, such as concurrent detection of cytoplasmic GFP and cell cycle.
Multi-pass flow cytometry reduces the barrier to high-marker analysis by simplifying complex panels and enabling detection of sensitive signals without compromise.
Supported by NIH grant R44GM139504
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a wide range of disease severity, ranging from asymptomatic infection to a life-threating illness, particularly in the elderly ...population and individuals with comorbid conditions. Among individuals with serious coronavirus 2019 (COVID-19) disease, acute respiratory distress syndrome (ARDS) is a common and often fatal presentation. Animal models of SARS-CoV-2 infection that manifest severe disease are needed to investigate the pathogenesis of COVID-19–induced ARDS and evaluate therapeutic strategies. We report two cases of ARDS in two aged African green monkeys (AGMs) infected with SARS-CoV-2 that had pathological lesions and disease similar to severe COVID-19 in humans. We also report a comparatively mild COVID-19 phenotype characterized by minor clinical, radiographic, and histopathologic changes in the two surviving, aged AGMs and four rhesus macaques (RMs) infected with SARS-CoV-2. Notable increases in circulating cytokines were observed in three of four infected, aged AGMs but not in infected RMs. All the AGMs had increased levels of plasma IL-6 compared with baseline, a predictive marker and presumptive therapeutic target in humans infected with SARS-CoV-2. Together, our results indicate that both RMs and AGMs are capable of modeling SARS-CoV-2 infection and suggest that aged AGMs may be useful for modeling severe disease manifestations, including ARDS.
Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further ...immunization, we demonstrate germinal centre B (B
) cells that last for at least 6 months. A 186-fold increase in B
cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of B
cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding B
cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells
. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous B
cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.
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