The present panel was optimized to quantify the relative frequencies of γδT-cells, invariant natural killer T-cells (iNKT-cells), and hematopoietic precursors in peripheral blood mononuclear cells (PBMC) from healthy individuals (Table 1). It works well with cryopreserved PBMC and we have observed similar results with fresh specimens. Other tissue types have not been tested. We developed this panel (Table 2) as part of a large study where we aim to survey the relative proportion of different immune cell subsets, including hematopoietic stem cells (HSC), in human peripheral blood specimens from healthy adults. It addresses HSC, γδT-cells, and iNKT-cells. HSC are multipotent precursor cells that give rise to all blood cell types, including the myeloid and lymphoid lineages. Though predominantly found in bone marrow and umbilical cord blood, they also occur at reduced frequencies in the blood 1, and can be identified by their expression of CD34 1, 2. In spite of being generally used as a molecular marker of HSCs, the function of CD34 is poorly understood 3. While most T-cells express a T-cell receptor (TCR) comprised of an α- and a β-chain, a minority of blood T-cells express the γδTCR. In healthy individuals, the vast majority of these have one of two phenotypes, representing ontologically separate lineages: DV1+ (previously Vδ1) cells are prevalent during fetal and early life, while DV2+ (previously Vδ2) cells usually dominate in adult blood 4, 5. The latter are usually GV9+ (previously Vγ9), but DV1 associates with a number of different Vγ chains 6. γδT-cells, in particular GV9/DV2 cells, are thought to act as a bridge between innate and acquired immunity 7. iNKT-cells express the AV24/BV11 TCR (previously Vα24/Vβ11) and recognize CD1d-restricted lipid antigens. The classical antigen used to detect these cells is the marine sponge-derived α-galactosylceramide (α-GalCer), though more common environmental Ags have recently been shown to also stimulate iNKT-cells 8, 9. CD1d molecules loaded with the α-GalCer analogue PBS-57 form more stable multimeric complexes than those loaded with α-GalCer, thus making a good tool to identify iNKT-cells 10. Three iNKT subsets have been characterized that differ in function, but also in CD4/CD8 expression: cytokine-producing CD4+ CD8− (predominant in fetal and neonatal blood), cytotoxic CD4− CD8−, and the rare IFN-γ-producing CD4− CD8+ iNKT-cells 11. Finally, we included Abs to CCR5, CCR7, CD27, CD28, and CD45RA in order to further explore the differentiation phenotypes of both γδT-cells and iNKT-cells (Figure 1). None to date. Additional and updated supporting information including technical details may be found in the online version of this article. Online Table 1 Instrument configuration. Online Table 2 Commercial reagents used in OMIP-019. Online Table 3 In-house conjugated reagents used in OMIP-019. Online Table 4 Priority rating for reagents. Online Table 5 Reagents tested but not included in final panel. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract Quantum dots (QD) are fluorescent nanocrystals that are highly useful in imaging and flow cytometric analyses. During routine use of monoclonal antibody conjugates of QD, we have occasionally seen partial or total loss of fluorescence when using certain lots of fixative solutions. We hypothesized that a low level contamination with heavy metal cations was responsible, since low level metal contaminants are not uncommon in formalin solutions. By titrating known concentrations of heavy metal cations into staining solutions, we found that millimolar concentrations of ferrous and zinc ions, and as low as 50 nanomolar cupric ions, completely eliminated QD fluorescence. By mass spectroscopic quantification of metals in commercial fixative solutions previously shown to perform poorly or well with regard to QD fluorescence, we confirmed that the presence of copper in solution was correlated with poor performance. Notably, prior addition of EDTA to chelate the divalent cations in these solutions prevented the inhibition of QD fluorescence. Finally, the copper‐induced loss of QD fluorescence is irreversible: cells labeled with QD are highly fluorescent and can be rendered nonfluorescent by the addition of cupric sulfate, even after washing extensively. Indeed, these cells can then be successfully stained with other QD reagents, providing a method for immunofluorescence restaining of cells without contaminating fluorescence from the first stain. Published 2010 Wiley‐Liss, Inc.
Background While several algorithms for the comparison of univariate distributions arising from flow cytometric analyses have been developed and studied for many years, algorithms for comparing multivariate distributions remain elusive. Such algorithms could be useful for comparing differences between samples based on several independent measurements, rather than differences based on any single measurement. It is conceivable that distributions could be completely distinct in multivariate space, but unresolvable in any combination of univariate histograms. Multivariate comparisons could also be useful for providing feedback about instrument stability, when only subtle changes in measurements are occurring. Methods We apply a variant of Probability Binning, described in the accompanying article, to multidimensional data. In this approach, hyper-rectangles of n dimensions (where n is the number of measurements being compared) comprise the bins used for the chi-squared statistic. These hyper-dimensional bins are constructed such that the control sample has the same number of events in each bin; the bins are then applied to the test samples for chi-squared calculations. Results Using a Monte-Carlo simulation, we determined the distribution of chi-squared values obtained by comparing sets of events from the same distribution; this distribution of chi-squared values was identical as for the univariate algorithm. Hence, the same formulae can be used to construct a metric, analogous to a t-score, that estimates the probability with which distributions are distinct. As for univariate comparisons, this metric scales with the difference between two distributions, and can be used to rank samples according to similarity to a control. We apply the algorithm to multivariate immunophenotyping data, and demonstrate that it can be used to discriminate distinct samples and to rank samples according to a biologically-meaningful difference. Conclusion Probability binning, as shown here, provides a useful metric for determining the probability with which two or more multivariate distributions represent distinct sets of data. The metric can be used to identify the similarity or dissimilarity of samples. Finally, as demonstrated in the accompanying paper, the algorithm can be used to gate on events in one sample that are different from a control sample, even if those events cannot be distinguished on the basis of any combination of univariate or bivariate displays. Cytometry 45:47–55, 2001. Published 2001 Wiley-Liss, Inc.
Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans.In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness.
