Leonore A. Herzenberg

Properties of Monoclonal Antibodies to Mouse Ig Allotypes, H-2, and Ia Antigens

Advances in somatic cell hybridization techniques have made it possible to generate hybrid cell lines producing monospecific antibodies directed at desired antigenic determinants (1). In this paper a modification of the cell fusion procedure (2,3) was used to recover stable hybrid cell lines secreting IgG antibodies to: (a) mouse major histocompatibility complex (MHC) alloantigens (H-2K and I-A); and (b) mouse immunoglobulin (Ig) allotypes (Ig-1b, Ig-5a, and Ig-5b).

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-

Fluorescence Activated Cell Sorting

An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

Increased production of IL-7 accompanies HIV-1–mediated T-cell depletion: implications for T-cell homeostasis

We hypothesized that HIV-1–mediated T-cell loss might induce the production of factors that are capable of stimulating lymphocyte development and expansion. Here we perform cross-sectional (n = 168) and longitudinal (n = 11) analyses showing that increased circulating levels of interleukin (IL)-7 are strongly associated with CD4+ T lymphopenia in HIV-1 disease. Using immunohistochemistry with quantitative image analysis, we demonstrate that IL-7 is produced by dendritic-like cells within peripheral lymphoid tissues and that IL-7 production by these cells is greatly increased in lymphocyte-depleted tissues. We propose that IL-7 production increases as part of a homeostatic response to T-cell depletion.

CD8 naive T cell counts decrease progressively in HIV-infected adults.

We show here that CD8 naive T cells are depleted during the asymptomatic stage of HIV infection. Although overall CD8 T cell numbers are increased during this stage, the naive CD8 T cells are progressively lost and fall in parallel with overall CD4 T cell counts. In addition, we show that naive CD4 T cells are preferentially lost as total CD4 cell counts fall. These findings, presented here for adults, and in the accompanying study for children, represent the first demonstration that HIV disease involves the loss of both CD4 T cells and CD8 T cells. Furthermore, they provide a new insight into the mechanisms underlying the immunodeficiency of HIV-infected individuals, since naive T cells are required for all new T cell-mediated immune responses. Studies presented here also show that the well-known increase in total CD8 counts in most HIV-infected individuals is primarily due to an expansion of memory cells. Thus, memory CD8 T cells comprise over 80% of the T cells in PBMC from individuals with < 200 CD4/microliter, whereas they comprise roughly 15% in uninfected individuals. Since the naive and memory subsets have very different functional activities, this altered naive/memory T cell representation has significant consequences for the interpretation of data from in vitro functional studies.

Inherent specificities in natural antibodies: a key to immune defense against pathogen invasion

Natural antibodies are produced at tightly regulated levels in the complete absence of external antigenic stimulation. They provide immediate, early and broad protection against pathogens, making them a crucial non-redundant component of the humoral immune system. These antibodies are produced mainly, if not exclusively, by a subset of long-lived, self-replenishing B cells termed B-1 cells. We argue here that the unique developmental pattern of these B-1 cells, which rests on positive selection by self antigens, ensures production of natural antibodies expressing evolutionarily important specificities that are required for the initial defense against invading pathogens. Positive selection for reactivity with self antigens could also result in the production of detrimental anti-self antibodies. However, B-1 cells have evolved a unique response pattern that minimizes the risk of autoimmunity. Although these cells respond rapidly and strongly to host-derived innate signals, such as cytokines, and to pathogen-encoded signals, such as lipopolysaccharide and phosphorylcholine, they respond very poorly to receptor-mediated activation. In addition, they rarely enter germinal centers and undergo affinity maturation. Thus, their potential for producing high-affinity antibodies with harmful anti-self specificity is highly restricted. The positive selection of B-1 cells occurs during the neonatal period, during which the long-lived self-renewing B-1 population is constituted. Many of these cells (B-1a) express CD5, although a smaller subset (B-1b) does not express this surface marker. Importantly, B-1a cells should not be confused with short-lived anergic B-2 cells, which originate in the bone marrow in adults and initiate CD5 expression and programmed cell death following self-antigen recognition. In summary, we argue here that the mechanisms that enable natural antibody production by B-1 cells reflect the humoral immune system, which has evolved in layers whose distinct developmental mechanisms generate complementary repertoires that collectively operate to maximize flexibility in responses to invading pathogens. B-2 cells, present in what may be the most highly evolved layer(s), express a repertoire that is explicitly selected against self recognition and directed towards the generation of high-affinity antibody response to external antigenic stimuli. B-1 cells, whose repertoire is selected by recognition of self antigen, belong to what may be earlier layer(s) and inherently maintain production of evolutionarily important antibody specificities that respond to pathogen-related, rather then antigen-specific signals.

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

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.

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.

Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.

We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen‐combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.