High-dimensional multi-pass flow cytometry via spectrally encoded cellular barcoding.

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.

Immunomodulatory potential of in vivo natural killer T (NKT) activation by NKTT320 in Mauritian-origin cynomolgus macaques.

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.

Blocking α4ß7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4ß7 with an anti-α4ß7 monoclonal antibody (Rh-α4ß7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4ß7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4ß7 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-α4ß7 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 α4ß7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

Declining neutrophil production despite increasing G-CSF levels is associated with chronic inflammation in elderly rhesus macaques.

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-1ß 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.