Emergence of multi-body interactions in a fermionic lattice clock.

Alkaline-earth atoms have metastable 'clock' states with minute-long optical lifetimes, high-spin nuclei and SU(N)-symmetric interactions, making them powerful platforms for atomic clocks1, quantum information processing2 and quantum simulation3. Few-particle systems of such atoms provide opportunities to observe the emergence of complex many-body phenomena with increasing system size4. Multi-body interactions among particles are emergent phenomena, which cannot be broken down into sums over underlying pairwise interactions. They could potentially be used to create exotic states of quantum matter5,6, but have yet to be explored in ultracold fermions. Here we create arrays of isolated few-body systems in an optical clock based on a three-dimensional lattice of fermionic 87Sr atoms. We use high-resolution clock spectroscopy to directly observe the onset of elastic and inelastic multi-body interactions among atoms. We measure the frequency shifts of the clock transition for varying numbers of atoms per lattice site, from n = 1 to n = 5, and observe nonlinear interaction shifts characteristic of elastic multi-body effects. These measurements, combined with theory, elucidate an emergence of SU(N)-symmetric multi-body interactions, which are unique to fermionic alkaline-earth atoms. To study inelastic multi-body effects, we use these frequency shifts to isolate n-occupied sites in the lattice and measure the corresponding lifetimes of the clock states. This allows us to access the short-range few-body physics without experiencing the systematic effects that are encountered in a bulk gas. The lifetimes that we measure in the isolated few-body systems agree very well with numerical predictions based on a simple model for the interatomic potential, suggesting a universality in ultracold collisions. By connecting these few-body systems through tunnelling, the favourable energy and timescales of the interactions will allow our system to be used for studies of high-spin quantum magnetism7,8 and the Kondo effect3,9.

A Fermi-degenerate three-dimensional optical lattice clock.

Strontium optical lattice clocks have the potential to simultaneously interrogate millions of atoms with a high spectroscopic quality factor of 4 × 1017 Previously, atomic interactions have forced a compromise between clock stability, which benefits from a large number of atoms, and accuracy, which suffers from density-dependent frequency shifts. Here we demonstrate a scalable solution that takes advantage of the high, correlated density of a degenerate Fermi gas in a three-dimensional (3D) optical lattice to guard against on-site interaction shifts. We show that contact interactions are resolved so that their contribution to clock shifts is orders of magnitude lower than in previous experiments. A synchronous clock comparison between two regions of the 3D lattice yields a measurement precision of 5 × 10-19 in 1 hour of averaging time.

Spin-orbit-coupled fermions in an optical lattice clock.

Engineered spin-orbit coupling (SOC) in cold-atom systems can enable the study of new synthetic materials and complex condensed matter phenomena. However, spontaneous emission in alkali-atom spin-orbit-coupled systems is hindered by heating, limiting the observation of many-body effects and motivating research into potential alternatives. Here we demonstrate that spin-orbit-coupled fermions can be engineered to occur naturally in a one-dimensional optical lattice clock. In contrast to previous SOC experiments, here the SOC is both generated and probed using a direct ultra-narrow optical clock transition between two electronic orbital states in 87Sr atoms. We use clock spectroscopy to prepare lattice band populations, internal electronic states and quasi-momenta, and to produce spin-orbit-coupled dynamics. The exceptionally long lifetime of the excited clock state (160 seconds) eliminates decoherence and atom loss from spontaneous emission at all relevant experimental timescales, allowing subsequent momentum- and spin-resolved in situ probing of the SOC band structure and eigenstates. We use these capabilities to study Bloch oscillations, spin-momentum locking and Van Hove singularities in the transition density of states. Our results lay the groundwork for using fermionic optical lattice clocks to probe new phases of matter.

Systematic evaluation of an atomic clock at 2 × 10(-18) total uncertainty.

The pursuit of better atomic clocks has advanced many research areas, providing better quantum state control, new insights in quantum science, tighter limits on fundamental constant variation and improved tests of relativity. The record for the best stability and accuracy is currently held by optical lattice clocks. Here we take an important step towards realizing the full potential of a many-particle clock with a state-of-the-art stable laser. Our (87)Sr optical lattice clock now achieves fractional stability of 2.2 × 10(-16) at 1 s. With this improved stability, we perform a new accuracy evaluation of our clock, reducing many systematic uncertainties that limited our previous measurements, such as those in the lattice ac Stark shift, the atoms' thermal environment and the atomic response to room-temperature blackbody radiation. Our combined measurements have reduced the total uncertainty of the JILA Sr clock to 2.1 × 10(-18) in fractional frequency units.

Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.

Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study.

Ibrutinib and other targeted inhibitors of B-cell receptor signaling achieve impressive clinical results for patients with chronic lymphocytic leukemia (CLL). A treatment-induced rise in absolute lymphocyte count (ALC) has emerged as a class effect of kinase inhibitors in CLL and warrants further investigation. Here we report correlative studies in 64 patients with CLL treated with ibrutinib. We quantified tumor burden in blood, lymph nodes (LNs), spleen and bone marrow, assessed phenotypic changes of circulating cells and measured whole-blood viscosity. With just one dose of ibrutinib, the average increase in ALC was 66%, and in>40% of patients the ALC peaked within 24 h of initiating treatment. Circulating CLL cells on day 2 showed increased Ki67 and CD38 expression, indicating an efflux of tumor cells from the tissue compartments into the blood. The kinetics and degree of the treatment-induced lymphocytosis was highly variable; interestingly, in patients with a high baseline ALC the relative increase was mild and resolution rapid. After two cycles of treatment the disease burden in the LN, bone marrow and spleen decreased irrespective of the relative change in ALC. Whole-blood viscosity was dependent on both ALC and hemoglobin. No adverse events were attributed to the lymphocytosis.

Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL.

Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

Human peripheral blood B-cell compartments: a crossroad in B-cell traffic.

A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues. These B-cell subpopulations, which are produced in the BM and lymphoid tissues, recirculate through peripheral blood (PB), into different tissues including mucosa and the BM, where long-living plasma cells produce antibodies. These circulating PB B-cells can be classified according to their maturation stage into i) immature/transitional, ii) naïve, and iii) memory B-lymphocytes, and iv) plasmablasts/plasma cells. Additionally, unique subsets of memory B-lymphocytes and plasmablasts/plasma cells can be identified based on their differential expression of unique Ig-heavy chain isotypes (e.g.: IgM, IgD, IgG, IgA). In the present paper, we review recent data reported in the literature about the distribution, immunophenotypic and functional characteristics of these cell subpopulations, as well as their distribution in PB according to age and seasonal changes. Additional information is also provided in this regard based on the study of a population-based cohort of 600 healthy adults aged from 20 to 80 years, recruited in the Salamanca area in western Spain. Detailed knowledge of the distribution and traffic of B-cell subsets through PB mirrors the immune status of an individual subject and it may also contribute to a better understanding of B-cell disorders related to B-cell biology and homeostasis, such as monoclonal B-cell lymphocytosis (MBL).

Discrepancy in measuring CD4 expression on T-lymphocytes using fluorescein conjugates in comparison with unimolar CD4-phycoerythrin conjugates.

BACKGROUND: Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROL Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. METHODS: Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. RESULTS: The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. CONCLUSION: CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards.

CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery.

BACKGROUND: CD69 is a surrogate marker of T-cell responsiveness to mitogen and Ag stimulus and can be used as a measure of T-lymphocyte activation. Quantitative flow cytometric determination of CD69 expression on T lymphocytes has several advantages over traditional lymphocyte proliferation assays, but this method has not yet been standardized for clinical applications. METHODS: We qualified a commercially available assay using the manufacturer's procedures for measurement of T-cell response to a mitogen (PHA), superantigen (Staphylococcus endotoxin B; SEB) and Ca(2+) ionophore (phorbyl myristate acetate; PMA) with peripheral blood from healthy volunteers. Following this, we tested the usefulness of the assay in determining T-cell responses to PHA and SEB for six immunocompromised patients. RESULTS: Healthy volunteers showed 17-fold increases in T-cell CD69 Ab bound per cell (ABC) with PHA stimulation compared with the baseline. SEB was also an effective T-cell activating agent, increasing CD69 ABC by 5-fold, comparable with results obtained with PMA stimulation. PHA- and SEB-stimulated T-cell CD69 ABC for patients 100 days post-BM transplant were generally below 1 SD of that from healthy volunteers. SEB-stimulated T-cell CD69 expression was significantly depressed for CD8(+) T cells while CD4(+) T-cell responses to SEB were generally within 1 SD of the mean for healthy volunteers. DISCUSSION: These results suggest that quantitative measurement of CD69 surface expression by flow cytometry is a useful diagnostic tool for detailed assessment of T-lymphocyte and subset activation.

Studies in NZB IL-10 knockout mice of the requirement of IL-10 for progression of B-cell lymphoma.

NZB mice develop an age-related malignant expansion of a subset of B cells, B-1 cells, with autocrine production of IL-10. IL-10, a pleiotropic cytokine with anti-inflammatory properties, is a potent growth and survival factor for malignant B cells. To further examine the in vivo requirement for IL-10 in the development and expansion of malignant B-1 clones in NZB mice, we developed a strain of homozygous IL-10 knockout (KO) mice on an NZB background. The NZB IL-10 KO mice develop peritoneal B-1 cells with approximately the same frequency as heterozygous and wild-type littermates. In contrast, the development of malignant B-1 cells in the peripheral blood and spleen, observed in wild-type NZB, rarely occurred in the NZB IL-10 KO. Phenotypic analysis of surface marker expression in splenic B cells indicated that, in contrast to the NZB with malignant B-1 splenic lymphoma, the surface marker expression of NZB IL-10 KO splenic B cells indicated that the majority of the B cells were typical B-2 cells. In the absence of IL-10, spontaneously activated B cells and antiapoptotic gene expression were reduced and lymphoma incidence was decreased. These results indicate that IL-10 is a critical factor for the progression of this B-cell malignant disease.

Clinical characteristics of familial B-CLL in the National Cancer Institute Familial Registry.

In an ongoing study, families with two or more living cases of B-CLL in first-degree relatives have been recruited through physician and self-referral. Since 1967, 28 kindreds with 73 cases of B-CLL have been enrolled within the National Cancer Institute (NCI) Familial B-CLL Registry. Medical, clinical, and demographic information have been obtained from private physicians, patient interview, hospital records, and death certificates. We used SEER Registry data to compare characteristics of sporadic B-CLL to familial B-CLL. The mean age at diagnosis was approximately 10 years younger among familial cases (57.9 +/- 12.1) than that observed in sporadic cases (70.1 +/- 11.9). A higher percentage of second primary tumors among familial CLL cases compared to reports in sporadic was also observed (16% vs. 8.8%). However, the transformation rate to non-Hodgkin's lymphoma does not appear to be different from that reported for sporadic cases. In conclusion, we observed some differences between familial and sporadic cases; whether any of these characteristics affect survival time or severity of disease is unknown. The study of families with multiple B-CLL cases will aid in delineating the genes and environmental factors that may play a role in the development of both forms of B-CLL.

Detection of unseparated human lymphocytes by flow cytometry.

The protocol for flow cytometry analysis presented here has been specifically developed for studies of human peripheral blood cells. In this protocol, analysis is performed on unseparated cells in whole peripheral blood, rather than on Ficoll-Hypaque-purified mononuclear cells. The advantage of this approach is that it requires less time, uses smaller blood volumes, and eliminates possible differential blood loss as a result of cell separation techniques. In this regard, B cell recovery using the whole blood method is significantly greater than that obtained using Ficoll-Hypaque-purified mononuclear cells. However, because lymphocytes generally represent a minority of peripheral cells (especially in adults), careful gating of the test samples for lymphocytes is a more critical requirement in this procedure than in other procedures using purified cells.

Diagnostic flow cytometry in hematologic malignancies.

During the past 30 years, we have seen flow cytometry (FCM) emerge from being a research tool requiring a group of engineers, an optical bench, and a darkened room to a benchtop flow cytometer that is used routinely in a clinical setting. The flow cytometer is to cell biology what the UV-visible spectrophotometer is to solution spectroscopy. Several events have contributed to the development of this technology to where it is now an indispensable tool in the diagnosis and clinical monitoring of disease. Many cell types are now under intense investigation. The clinical application of FCM to lymphocyte subset immunophenotyping in the leukemias and lymphomas was responsible for the early development of clinical FCM.

Quantitative fluorescence cytometry.

The phenotypes useful in distinguishing normal and neoplastic leukocytes are often identified by fluorescence staining reactions detected on flow cytometers. These reactions were originally observed by fluorescence microscopy, and cells were classified by human observers as simply negative or positive, with the positive cells sometimes distinguished as dim or bright. These terms are still used in analyzing flow cytometry (FCM) results. However, recent advances in our understanding of fluorescence signals from stained cells (1) now permit the translation of terms like "dim" and "bright" into real mass units of fluorescence intensity, a process that we call quantitative fluorescence cytometry (QFCM). Although the translation is not yet exact and certain technical details remain to be resolved, a general understanding of QFCM is now accessible and helpful in interpreting staining patterns.