Leonard A. Herzenberg

Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens.

Xenogeneic immunizations have the advantage of detecting a wide range of antigenic determinants because many commonly occurring proteins have diverged significantly during the course of evolution and are thus antigenic in other species. The broadness of xenogeneic responses, however, means that the antisera they produce are usually complex and require extensive absorptions to make them specific for a single antigen. This problem has now been overcome by generating hybridomas producing monoclonal antibodies (Kohler & Milstein 1975). These permit dissection ofthe xenogeneic response so that large amounts of individual antibodies can be obtained, each of which recognizes only one of the determinants recognized by a broadly reactive conventional antiserum. Williams et al. (1977) used hybridoma monoclonal antibodies obtained after immunizations of mice with rat cells to study rat cell-surface antigens present on subpopulations of rat lymphocytes, i.e., differentiation antigens. Springer et al. (1978a) and Stern et al. (1978) used a similar approach to study mouse lymphocyte antigens. They prepared monoclonal antibodies by immunizing rats with mouse lymphocytes and showed that these monoclonals recognized previously undetected mouse cell surface determinants including a glycoprotein antigen that appears to be specific for macrophages (Springer et al. 1978b). Trowbridge (1978) also used rat anti-mouse immunizations to generate a monoclonal antibody against the non-polymorphic lymphocyte surface antigen T200.

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-

11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity.

11-color, 13-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity

Analysis of cell populations with a fluorescence-activated cell sorter.

Cell biologists have long been concerned with morphologic and functional characterization of the different cell types present in various tissues. Immunofluorescence has been applied t o this problem t o differentiate cell populations according t o their various antigenic characteristics. Visual methods of detecting the fluorescence have limitations, because classification of a cell as fluorescent or nonfluorescent can be subjective. In addition, fluorescence microscopy is not readily adapted to the quantitative analysis of fluorochromes on different cells. A fluorescence-activated cell sorter (FACS), designed to characterize and isolate viable cells, can be used to obviate some of the analytic difficulties encountered with visual methods. The FACS analyzes a cell preparation with respect t o two parameters in a quantitative rather than qualitative manner. It also permits physical separation of these cells for further study. Individual viable cells, stained by conventional irnmunofluorescence techniques, are analyzed for both fluorescence and low-angle light scattering as they pass through a laser beam. The frequency distributions of these two parameters are used to characterize the cell population. The criterion for denoting a specific subpopulation is obtained from these histograms, so that the classification of cells into one or another category is more objective compared with visual techniques. Subpopulations of cells identified by this type of analysis can then be isolated with the FACS and assayed for various functional activities.

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.

Cell Sorting: Automated Separation of Mammalian Cells as a Function of Intracellular Fluorescence

A system for high-speed sorting of fluorescent cells was able to sort mouse spleen cells from Chinese hamster ovarian cells after development of fluorochromasia. Highly fluorescent fractions separated after similar treatment from mouse spleen cells immunized to sheep erythrocytes were enriched in antibody-producing cells by factors of 4 to 10.

The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes.

Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF‐AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c‐fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors.

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets

The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.

Toward a layered immune system

Leonore A. Hetzenberg and Leonard A. Herzenberg Department of Genetics Stanford University School of Medicine Stanford, California 94305 The broad outlines of developmental relationships among cells in the immune system appeared to be established some years ago with the introduction of the idea that pluripotent hematopoietic stem cells emerge early in fetal life and persist as a self-replenishing population that con- tinues to divide and differentiate throughout life without changing its original potential. Recent studies of lympho- cyte development challenge this paradigm (Herzenberg and Stall, 1989; Linton et al., 1989), suggesting that there are instead several types of hematopoietic stem cells that have evolved sequentially and function at specified times during development; these create layers of progressively more advanced populations of lymphocytes and myeloid cells that collectively provide the diverse capabilities of the mammalian immune system. According to previous dogma, the self-replenishing he- matopoietic stem cells that reside initially in fetal liver and then in adult spleen and bone marrow provide a continu- ing source of myeloid and lymphoid stem cells. These my- eloid and lymphoid stem cell populations are themselves self-replenishing and collectively give rise to all erythro- cytes, myelocytes (granulocytes, macrophages, etc.), and lymphocytes found in the fetus, neonate, and adult. Thus, cells in the hematopoietic system are viewed as a single lineage that is derived from a single progenitor popula- tion, and lymphocytes are one of two major subdivisions of that lineage. The origin of various types of 6 and T lymphocytes can be incorporated into an extension of this model that in- troduces further branchpoints in the overall lineage-one that separates developmental pathways for B cells and T cells, and others that distinguish pathways for various subsets of T and B cells. Some workers refer to each of these pathways as a separate lineage; others reserve the term lineage for those pathways that appear to originate from a distinct progenitor with (at least a limited) capacity for self-regeneration and the ability to give rise to the later cells in the pathway. However, all agree that lymphocyte differentiation proceeds along pathways that at some level merit the designation lineages, because lymphocytes ori- ginate with distinct progenitors that divide and differenti- ate to populate sequential stages of the pathway. The B cell lineages proposed by Klinman and col- leagues (Linton et al., 1989) fall within this general frame- work. The existence of these lineages is predicated on evi- dence indicating that, contrary to conventional textbook wisdom, virgin B cells participating in primary antibody re- sponses are developmentally distinct from memory B cells that participate in secondary antibody responses to the same antigens. Some questions can be raised con- cerning the generality of these findings and their interpre-

Two-color immunofluorescence using a fluorescence-activated cell sorter.:

A technique for the analysis of fluorescein and rhodamine in a flow system using a single wavelength of excitation is described. Both optics and electronics are used to discriminate the fluorescein and rhodamine signals. This technique has been used to study the relationship between immunoglobulin M and immunoglobulin D on mouse splenic lymphocytes.