David R. Parks

The LY-1B Cell Lineage

The murine Ly-I lymphocyte surface glycoprotein was defined initially with conventional antisera in cytotoxic assays (Cantor & Boyse 1977). As such, it appeared to be expressed exclusively on helper T cells (Cantor & Boyse 1975). Later, however. Fluorescence Activated Cell Sorter (FACS) analyses and sorting studies with monoclonal antibody reagents showed that all T cells express Ly-1, regardless of functional subclass (Ledbetter et al. 1980). Furthermore, these studies (Lanier et al. 1981a, 1981b) showed that Ly-1 is expressed on several murine B cell tumors and introduced evidence suggesting that this glycoprotein may also expressed on a small proportion of normal murine splenic B cells (Manohar et al. 1982, Hayakawa et al. 1983). Similar studies with human lymphocytes demonstrated the homologous {LeuI) cell surface antigen on all normal T cells (Ledbetter et al. 1981), on some B cell tumors (particularly chronic lymphocytic leukemias) (Martin et al. 1981) and, as in the mouse, on a small proportion of apparently normal B cells (CalligarisCappio et al. 1982). Thus, a series of earlier findings foreshadowed contemporary evidence demonstrating Ly-I and Leu-1, respectively, on subsets of murine and human B cells and showing further that Ly-1 marks functionally distinct B cells that play a major role in autoimmunity in the mouse. In this paper, we summarize the physical and functional characteristics that distinguish Ly-1 B cells from the majority of splenic and lymph node (conventional) B cells. We focus on data from cell transfer and antibody treatment studies, which locate Ly-I B cells in a separate developmental lineage that branches off from the conventional lymphocyte developmental lineage during prenatal or early neonatal life. We then consider various genetic defects that influence autoantibody production and Ly-I B representation and, finally, we discuss potential homolog-

Interpreting flow cytometry data: a guide for the perplexed

Recent advances in flow cytometry technologies are changing how researchers collect, look at and present their data.

Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene.

The nef gene, which encodes related cytoplasmic proteins in both human (HIV) and simian (SIV) immunodeficiency viruses is dispensable for viral replication in vitro. In contrast, in vivo experiments have revealed that SIV nef is required for efficient viral replication and development of AIDS in SIV infected rhesus monkeys, thus indicating that nef plays an essential role in the natural infection. We show that expression of the Nef protein from the HIV‐1 NL43 isolate in transgenic mice perturbs development of CD4+ T cells in the thymus and elicits depletion of peripheral CD4+ T cells. Thymic T cells expressing NL43 Nef show altered activation responses. In contrast, Nef protein of the HIV‐1 HxB3 isolate does not have an overt effect on T cells when expressed in transgenic animals. The differential effects of the two HIV‐1 nef alleles in transgenic mice correlate with down‐regulation of CD4 antigen expression on thymic T cells. The differential interactions of the NL43 and HxB3 nef alleles with CD4 were reproduced in a transient assay in human CD4+ CEM T cells. Down‐regulation of CD4 by nef in both human and transgenic murine T cells indicates that the relevant interactions are conserved in these two systems and suggests that the consequences of Nef expression on the host cell function can be analyzed in vivo in the murine system. Our observations from transgenic mice suggest that nef‐elicited perturbations in T cell signalling play an important role in the viral life cycle in vivo, perhaps resulting in elimination of infected CD4+ T cells.

N-acetylcysteine replenishes glutathione in HIV infection.

Glutathione (GSH) deficiency is common in HIV‐infected individuals and is associated with impaired T cell function and impaired survival. N‐acetylcysteine (NAC) is used to replenish GSH that has been depleted by acetaminophen overdose. Studies here test oral administration of NAC for safe and effective GSH replenishment in HIV infection.

8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity.

We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems.

A new "Logicle" display method avoids deceptive effects of logarithmic scaling for low signals and compensated data.

In immunofluorescence measurements and most other flow cytometry applications, fluorescence signals of interest can range down to essentially zero. After fluorescence compensation, some cell populations will have low means and include events with negative data values. Logarithmic presentation has been very useful in providing informative displays of wide‐ranging flow cytometry data, but it fails to adequately display cell populations with low means and high variances and, in particular, offers no way to include negative data values. This has led to a great deal of difficulty in interpreting and understanding flow cytometry data, has often resulted in incorrect delineation of cell populations, and has led many people to question the correctness of compensation computations that were, in fact, correct.

Isolation of human myoblasts with the fluorescence-activated cell sorter

We have established procedures for the rapid and efficient purification of human myoblasts using the fluorescence-activated cell sorter. Our approach capitalizes on the specific reaction of monoclonal antibody 5.1H11 with a human muscle cell surface antigen. For each of the five samples analyzed, an enrichment of myoblasts to greater than 99% of the cell population was immediately achieved. Following 3 to 4 weeks of additional growth in vitro, sorted myoblast cultures remained 97% pure. Differentiation of the sorted myoblast cultures, assessed by creatine kinase activity and isozyme content, was comparable to that of pure myoblast cultures obtained by cloning, and was significantly greater than that of mixed fibroblast and myoblast cultures. An average of 10(4) viable myoblasts can be obtained per 0.1 g tissue, each with the potential to undergo approximately 40 cell divisions. Accordingly, if only two-thirds of this proliferative capacity is utilized, the potential yield approximates 10(12) myoblasts, equivalent to 1 kg of cells. Human myogenesis in vitro is no longer limited by cell number and is now amenable to molecular and biochemical analysis on a large scale.+

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

BACKGROUND This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.

Detection of fetal erythrocytes in maternal blood post partum with the fluorescence-activated cell sorter

A study was made of the frequency and amount of fetal hemorrhage into maternal blood during labor and delivery as evidenced by the number of fetal cells present in the maternal circulation immediately after spontaneous vaginal delivery. A sensitive, indirect immunofluorescence was used with fluorescence-activated cell sorter analysis of erythrocytes. All of the 16 Rh-negative mothers studied after vaginal delivery of Rh-positive infants had circulating Rh-positive cells. The mean Rh-positive to Rh-negative erythrocyte ratio was 1:14, 100 in maternal blood, which corresponds to a mean fetal hemorrhage of 156 microliters. The test described is sufficiently sensitive to be used for the study of primary Rh isoimmunization and could be clinically applicable for antepartum screening to determine which patients require Rh immune globulin treatment before delivery.

Dual immunofluorescence — new frontiers in cell analysis and sorting

Investigations of the nature and functions of the immune system and its cell populations have been revolutionized by the techniques of fluorescence-activated cell sorting (FA CS) and flow cytometry. Over the last few years, however, it has become increasingly clear that making only a single immunofluorescence measurement on each cell is not adequate for many investigations of lymphoid cell subpopulations. In this review the authors discuss why adequate resolution of functional subsets will increasingly require multiparameter definition including measurements on two or more fluorescent labels.