Human Lymphocyte Subpopulations1

Human Lymphocyte Su bpopu lations' 1. CHESS' AND S. F. SCHLOSSMAN Division of Tumor Immnology, Sidney F a h e r Concur Institute, Hamrrd M c a l school, Barton. MasnrdruseUs

1. Introduction

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11. Classical Cell Surface Determinants on Human Lymphocytes

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111. Antigens Distinguishing Human Thymocytes (HTL) and Peripheral Blood T-cell Subclasses (THI) ........................................... IV. Human B-Cell Specific Antigens .............................. V. Purification of Lymphocyte Subclasses ................................... A. Affinity Chromatography on Columns ................................ B. Fluorescence-Activated Cell Sorting ........... C. Rosette Depletion Techniques ....................................... D. Other Separation Techniques ....... ............................. VI. The Functional Analysis of Isolated Human Lymphocyte Subpopulations . . A. General Considerations .............................................. B. Proliferation in Response to Soluble and Cellular Antigens ............ C. Mediator Production by Subclasses of Human Lymphocytes ........... D. Cell-Mediated Lympholysis .................................. : ...... E. Cell-Mediated Destruction of Syngeneic Tumor Cells ................. F. Analysis of Human Peripheral Blood B-Cell Function ................. G. Characterization and Isolation of Regulator Cells in Human Peripheral Blood.. .................................................. H. The Functional Heterogeneity of Null Cells ............. References .............................................................

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I. Introduction

The precise dissection of the cellular mechanisms and interactions involved in the generation of the human immune response has been faciliated b y recent advances in three interrelated areas: (1) the development of in uitro methods for the characterization and identification of human lymphocyte classes by cell surface markers; (2) the development of new techniques for the isolation of highly purified subclasses of human lymphocytes and monocytes; and (3) the development of in uitro techniques to discriminate the functional properties and interactions of the isolated subsets of lymphocytes. These advances have occurred during the last few years despite the obvious limitations inherent in the study of the human immune re-

' This work was supported in part by A1 12069, CA-19589, N01-CB-43964, and N01CB-53881 from the National Institutes of Health. Present address: Dept. of Medicine, Columbia University College of Physicians and Surgeons, New York,New York. 2 13

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L. CHESS AND S. F. SCHLOSSMAN

sponse. In particular, one limitation has been the lack of genetically defined strains that have been so important in both the generation of alloantisera to unique subclasses of cells and in the genetic analysis of T, B, and macrophage cell interactions. In addition, in uivo studies of human immune finction are necessarily limited and have relied for the most part upon the direct in uitro and in uiuo study ofpatients with “ experiments of nature” that arise in the congenitally immunodeficient patient or in patients with autoimmunity. Even these studies have been limited both by the rarity and heterogeneity of the individual disorders. In the present review we focus our attention on some approaches to overcoming difficulties inherent in the investigation of the human system with particular attention to the three areas noted above. Emphasis is placed on studies undertaken in the author’s laboratory over the past 4 years, and no attempt is made to cite exhaustively many excellent studies carried out elsewhere using similar approaches. II. Classical Cell Surface Determinants on Human lymphocytes

During the early 1970s enormous excitement was generated in clinical immunology by the finding that human T and B cells could be readily distinguished by cell surface markers. These surface structures initially included intrinsically bound surface membrane Ig (Froland and Natvig, 1970; Grey et al., 1971; Siegal et al., 1971), the receptor for sheep erythrocytes ( E receptor) (Brain et al., 1970; Coombs et al., 1970; Lay et al., 1971; Jondal et al., 1972), receptors for the complement components (Bianco et al., 1970), and the receptors for the F c fragment of antibody molecules (Dickler and Kunkel, 1972; Basten et al., 1972). Many studies indicated that the subset of human lymphocytes forming rosettes with sheep erythrocytes (E+) were T cells (Froland, 1972; Jondal et al., 1972; Wybran et al., 1972). The evidence for this conclusion stemmed from studies demonstrating that E+ cells represent a population distinct from B lymphocytes as identified b y membrane-bound Ig; patients with profound hypogammaglobulinemia and B-cell deficiency have normal or increased numbers of E-rosetting cells; most human thymocytes are E rosette positive; and, few E-rosetting cells are found in patients with congenital thymic aplasia. In contrast, a reciprocal subset of human B cells was defined, which, like their counterparts in rodents, had intrinsically bound surface Ig and contained the receptor for the complement components C3d and C3b (see Moller, 1973). To support this view, it was shown that patients with infantile X-linked agammaglobulinemia lacked cells bearing surface Ig, whereas patients with congenital thymic ap-

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lasia had normal or higher percentages of Ig+ cells (Grey et al., 1971; Cooper et al., 1971; Geha et al., 1973). More recently, and as will be discussed in detail below, the evidence for the E-rosette marker as a distinguishing characteristic of human T cells and the evidence for the Ig and C receptor determinants for a distinguishing characteristic of B cells have been shown more directly in in vitro functional studies of isolated populations of T and B cells. With respect to the Fc receptor, initial studies indicated that the predominant cells bearing receptors for IgG in the human peripheral blood were surface Ig-bearing B lymphocytes and monocytes. Recent studies now indicate that a significant percentage of human T cells have receptors for the Fc fragment of IgG, and an even larger percentage have receptors for the F c fragment of IgM (Moretta et al., 1976, 1977). In addition, there exists a third population of cells in peripheral blood which, for want of a better term, have been referred to as a null-cell population (Jondal et al., 1973b; Greenberg et al., 1973; Chess et aZ., 1974~).This population of cells is E rosette negative and surface Ig negative, but heterogeneous with respect to both complement and Fc receptors. The subset of null cells bearing both the complement and Fc receptors has been isolated (see below) and shown to be the predominant lymphocyte effecting antibodydependent cellular cytotoxicity (Perlmann et al., 1975; MacDermott et al., 1975; Brier et al., 1975). The functional properties of Fc-bearing cells, which are E rosette negative, Ig negative, and lack complement receptors, are currently under active study. Taken together, the evidence suggests that the Fc receptor, although perhaps useful in the discrimination of subclasses of T or B cells, is not particularly useful for the initial characterization of human lymphocytes into T or B subpopulations. In addition to the four markers discussed above, a number of investigators have reported additional determinants that may distinguish human T and B cells. Thus, human T cells have been shown to form rosettes with rhesus monkey red cells (Lohrmann and Novikovs, 1974), human red cells (Kaplan and Clark, 1974), and with human B lymphoblasts (Jondal et al., 1975). In addition, they may contain receptors for measles-induced surface antigens (Valdimmarsson et al., 1975) and lectins such as the Helix pomatia ones (Hammarstrom et al., 1973). In contrast, human peripheral B cells have receptors for EB viral antigens (Jondal and Klein, 1973) and have been reported to have receptors for mouse red blood cells. These additional cell surface markers of human lymphocytes have been recently reviewed (Bloom and David, 1976). Since they have been less extensively analyzed then the four classic determinants, there is less unanimity on the proportion

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of cells bearing either of these determinants and, in fact, evidence still is sparse with respect to functional studies. As suggested above, the ability to characterize distinct subpopulations with the conventional techniques has allowed for their quantitation in peripheral blood and in other lymphoid tissue in both normal individuals and patients. Moreover, these cell surface markers have permitted the development of methods that allow for the isolation of distinct receptor-bearing lymphocyte populations for functional studies. Despite the usefulness of conventional cell surface markers, it should be emphasized that they are still relatively crude and inexact. In particular, the E- and EAC-rosetting tests have been difficult to quantitate and the results vary because of a number of technical factors; these include serum source, contamination with red cells, incubation temperature, and either the method of contrifugation or the force used in resuspension of the rosettes. In addition, the analysis of surface Ig on human B cells is complicated by the fact that B cells and other cell populations bear receptors for the Fc portion of immunoglobulins. Therefore, it has been emphasized that techniques involving identification of surface immunoglobulins with fhoresceinated anti-Ig reagents require prior pepsin digestion (Winchester et al., 197513). The analysis of Fc receptors on lymphoid cells depends on rosetting techniques using IgG- and IgM-coated red cells, binding of labeled aggregated IgC or antigen-antibody complexes. These methods have also been fraught with technical difficulties that have not as yet been completely resolved (Arbeit et aZ., 1976).The finding now of Fc receptors on human T cells adds considerably to these difficulties. Perhaps of greater biological importance, the techniques outlined above do not adequately deal with the extraordinary heterogeneity that is known to exist within the T, B, and null-cell populations. Dissection of this heterogeneity is critical for studies directed at the h n c tional properties of lymphocytes and for an understanding of the maturation and differentiation of human lymphocytes. For these reasons, considerable attention has been directed at defining more precise quantifiable surface determinants on human lymphocyte subclasses and relating these determinants to states of differentiation and functional properties of cells. 111. Antigens Distinguishing Human Thymocytes (HTL) and Peripheral Blood T-cell

Subclasses (THJ

In mice, the relationship between thymocytes and T-cell surface antigens with the distribution, life history, stages of differentiation, and, perhaps most interestingly, function of T-cell subclasses have

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been extensively analyzed (Shiku et al., 1975; Cantor and Boyse, 1975a,b; Cantor and Weissman, 1976). These phenotypic deterininants on the surface of thymus-derived cells for the most part are recognized by alloantisera prepared in mice bearing the appropriate genotypes (Shen et al., 1975). Whereas murine alloantisera to theta, TL, Lyl, Ly2, Ly3, and 1 region deteiminants have proved to b e extremely valuable, comparable reagents for human cells are just becoming available. For example, heteroantisera directed toward human T-cell determinants have been prepared using a variety of immunization schedules, absorption procedures, and methods for assay. The antigens used for preparing these antisera have included human thymocytes, soluble extracts of thymocytes, brain, leukemic lymphoblasts bearing E-rosette markers, peripheral lymphocytes from patients with X-linked agammaglobulinemia, and, recently, monkey thymocytes (Aiuti and Wigzell, 1973; Brown and Greaves, 1974; Greaves and Janossy, 1976; Balch et ul., 1976).Virtually all these heteroantisera require extensive absorption with combinations of cells including allogeneic cultured B-cell lymphoblastoid lines, B-cell chronic lymphatic leukemias, bone marrow cells, erythrocytes, and fetal cells to render them specific. Most of the heterologous antihuman T-cell reagents prepared to date d o not discriminate between thymocytes and peripheral T cells. In addition, many of the anti-T-cell sera are specific only by complement-dependent lytic assays, but not when analyzed by immunofluorescence, especially with extremely sensitive fluorescence techniques. In addition, only a few ofthe resulting anti-T-cell reagents appear to discriminate functionally distinct T-cell subclasses (Woody e t al., 1975; Brouet and Tobin, 1976). In an attempt to overcome some of these difficulties, w e have prepared antisera to highly purified T-cell and/or thymocyte populations in rabbits and absorbed the resulting antilymphocyte sera with autologous lymphoblastoid cell lines (Schlossman et al., 1976; Chess and Schlossman, 1977). We have found that the use of autologous B lymphoblastoid cell lines for absorption of heteroantisera, as well as the use of purified populations of T cells and thymocytes for immunization, are of considerable importance with respect t o the development of specific antibodies with a high degree of specificity. These approaches have facilitated the removal of species, differentiation, MHC or fetal antigens to which heteroantisera are often directed. For example, several antisera have been prepared that identify unique cell surface determinants on human thymocytes, but are not detected on more mature normal human peripheral T cells or on B cells. These antisera (anti-HTL) are prepared by immunizing rabbits with E-rosette-positive, acute lymphoblastic leukemic cells and absorbing

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the antisera with autologous B lymphoblastoid cell lines derived from the same patient when in clinical remission. The HTL antisera react exclusively with thymocytes and with leukemic cells from those patients with the T-cell variety of acute lymphoblastic leukemia (ALL). Since the anti-HTL sera react only with thymocytes and T-cell ALLs, they are analogous to the geneticaIly defined anti-TL sera developed in the mouse. Of importance is the failure to detect HTL antigens on mature normal peripheral T cells, which suggests that these antisera define a state of differentiation within the T-cell pool. Interestingly, the subgroup of ALL patients whose blast cells bear the HTL antigen frequently present with high white counts and thymic masses, and do poorly clinically with respect to chemotherapy (Sallan et al., 1977). The HTL unreactive patients, on the other hand, which account for approximately 80% of ALLs in childhood, bear Ia-like determinants on their blast cell surfaces (see below). Of additional interest is the fact that some of these anti-HTL antisera prepared by immunizing rabbits with T-cell leukemic blasts can be absorbed with thymocytes and subsequently shown to react with leukemic blast cells, but not with either thymocytes or purified T cells (unpublished observations). Although the HTL antiserum defines differentiation antigen(s) on thymocytes, its failure to react with peripheral T cells precludes its usefulness in distinguishing functionally unique subclasses of peripheral T cells. In order to develop antisera capable of distinguishing antigens on more differentiated T cells, we have used a similar approach to that outlined above. Highly purified T cells were isolated by anti-F(ab)*immunoabsorbent column chromatography, nylon passage, and rosette depletion of complement-bearing cells prior to immunization of rabbits. The resulting rabbit antisera was then absorbed with an autologous B-cell line (Evans et al., 1977).Again the use of the autologous B-cell line was shown to be more efficient for absorption of these antisera than were allogeneic B cell lines or chronic lymphocytic leukemia (CLL) cells. For example, antisera can be rendered T-cell specific by absorption with as few as 2 x 1W autologous B-lymphoblastoid cells. Their activity on peripheral T cells and thyrnocytes plateaus generally at 60 and 90%, respectively, and cannot be hrther absorbed by as many as 1 x lo9 B cells. At maximal concentrations of antisera, 5040% of purified T cells are lysed in the presence of complement. In contrast, 90-100% of thymocytes are lysed (Fig. 1). No further increase in T-cell lysis is obtained b y a second treatment with antibody and complement, suggesting that a discrete subclass of T cells is recognized. The antigen(s) recognized on T cells is designated THI,and the absorbed heteroantiserum as anti-TH1.Simi-

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lo 0

-

Ill60

l/aO

-

1/40

., 1/20

1/10

SERUM MLUTW

.,

FIG.1. Complement-dependent lysis of lymphoid subpopulations using serial dilutions of anti-THIabsorbed with 1 x 10” autologous lymphoblastoid cells. Fetal thymocytes; 0, peripheral T cells; 0 , unfractionatedlymphocytes; 0 , peripheral B cells.

lar results were obtained with anti-T,, serum when analyzed by immunofluorescence using a sandwhich technique with fluoresceinated goat) antirabbit F(ab)* on the fluorescence-activated cell sorter. Of more importance, as will be shown below, the anti-TH1serum identifies a functionally distinct subclass of human T cells. Taken together, the HTL and TH1 antigens allow a preliminary view of the differentiation of human T cells from thymocytes (Fig. 2). Thus, thymocytes bear receptors for sheep erythrocytes, HTL, and TH1antigens. With further differentiation, the sheep cell receptor is retained, whereas the HTL antigen is lost. In contrast, the TH1 antigen, which is detected on all thymocytes, is found in only 40-60% of peripheral T cells. Further DIFFERENTIATION ANTIGENS ON HUMAN T CELLS

FIG. 2. DiEerentiation antigens on human thymus-derived lymphoid cells.

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support for this differentiation scheme was obtained by analysis of T leukemic cells. In the childhood T-cell leukemias, the lymphoblasts are usually E rosette positive, HTL positive, and T H , negative. In contrast, the T lymphoblasts found in a variant to chronic lymphatic leukemia are E rosette positive, HTL negative, but bear the T H 1 antigen. Thus, the morphologically more mature leukemic cells in CLL, like the more differentiated T cells in normal individuals appear to have lost the HTL antigen, while maintaining the T H 1 antigen. We have not as yet identified an E rosette positive, HTL negative, TH1 negative subclass in chronic lymphatic leukemia, but one would predict that such leukemic cells, exist. IV. Human 8-Cell Specific Antigens

As indicated above, a considerable number of cell surface determinants have been used in defining and quantitating circulating human B cells. These include surface Ig, the complement receptor, the Fc receptor, EB viral receptors, and heteroantisera directed at B-lymphocyte antigens. Recently, a new group of polymorphic antigens has been characterized which are linked to the major histocompatibility locus and are expressed on B cells and monocytes, but not on T cells (van Rood et al., 1975; Jones et al., 1975; Mann et aZ., 1975; Winchester et al., 1975a). These groups of antigens are distinct from the gene products of the HLA-A, B, and C locus, which are expressed on essentially all types of cells with the exception of erythrocytes. The major histocompatibility complex (MHC) gene products expressed predominantly on B cells are closely linked to and include at least some of the HLA-D region determinants, which control the mixed lymphocyte reaction (MLR). It is of interest that these B cell determinants appear to be analogous to the murine Ia antigens even though they are found outside the HLA-A and HLA-B regions, whereas in the mouse, the genes controlling the expression of MLC and Ia antigenic determinants are mapped between the H2-D and H-2K loci. The human Ia-like determinants were first detected using human alloantibody obtained from multiparous females which had been absorbed with platelets to remove antibody directed toward HLA-A, B, and C gene products. Furthermore, Ia-like molecules of 23,000 and 30,000 molecular weight (p23,30) have now been isolated and purified from human B lymphoblastoid cell membranes and used to generate rabbit anti-p23,30 antibody (Humphreys et aZ., 1976). The p23,30 antigen was shown to be a B lymphocyte-specific cell surface polypeptide complex and immunochemically different from HLA-A,

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B, and C antigens, immunoglobulins, and p2 microglobulins. In addition, the antigen complex was shown to be easily identified on the surface of B lymphocytes and a subclass of null lymphocytes which bear the receptor for complement. The evidence that the p23,30 antigen complex is analogous to murine Ia antigens includes (a) chemical and structural similarities; (b) tissue distribution; (c) linkage to the MHC; and (d) biological function as judged b y effects of antisera to p23,30 in a variety of systems including the inhibition of the MLC reactivity and ADCC as well as its effects on the differentiation of B cells triggered b y products of activated human T cells (Strominger et al., 1976; Humphreys et al., 1976; Chess et al., 1976; Friedman et aZ.,

1977).

Similar Ia-like molecules have been isolated from other human B-cell lines. For example, an antigen complex very similar to the p23,30 antigen was isolated from the Yoder cell line, which is homozygous for HLA-2 and -7 (Fuks et d.,1977).The isolated p23,30 complex from these cells inhibits the cytotoxicity of B-cell alloantisera specific for Yoder cells while not inhibiting anti-HLA-2 or -7 sera. More recently, a similar Ia-like complex has been derived from the cultured human lymphoblastoid cell line BRI-8 (Snary et al., 1977). These antigens comprise two glycoproteins of 33,000 and 28,000 molecular weight and are therefore very similar to the p23,30 molecular complex derived from the IM-1 and Yoder cell lines. Antibody to these determinants blocks the MLC reaction, and the isolated antigens inhibit specifically B-cell alloantisera. Recently, we found b y complement-dependent lysis studies and by immunofluorescence that the p23,30 antigen is detected on a hnctionally important subclass of human monocytes (Breard et al., 1977) and, perhaps more interestingly, appears on highly purified populations of T cells after in uitro sensitization to alloantigen (Evans et aZ., 1977). These latter results suggest that Ia-like determinants may be masked and expressed on distinct subsets of human T cells and detected only by functional studies directed at the inhibition of selected T-cell functions. Alternatively, the expression of Ia on T cells may result from the binding of Ia-like molecules to putative Ia receptors on the surface of activated T cells. In Table I we have summarized the cell surface markers of human peripheral T, B, and null lymphocyte classes and thymocytes with respect to the conventional and more recently described cell surface determinants. These surface antigens provide the framework by which methods have been developed for the isolation and functional characterization of distinct human subclasses as described in the sections

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L. CHESS AND S. F. SCHLOSSMAN TABLE I SURFACE PROPERTIES OF HWAN PEIUPHERAL LYMPHOCYTE SUBSETSAND THYMOCYTES

Lymphocyte subpopulations

Surface determinants

E rosette

EAC rosette

SmIg

HTL

TI,

Ia-like (~23,301

T

B Null Thymocyte " Null cells are heterogeneous with respect to EAC receptors and p23,30, the same 2 0 3 0 % of cells reacting with each. 'ITH, is present on 50-60% of peripheral T cells and approximately 90% of th ymocytes. ' T cells activated by alloantigen express the p,23,30 antigen.

below. We would emphasize that, given the overlap between surface determinants on various subclasses of cells, it is important to analyze lymphocyte subpopulations not only by their surface determinants, but, in addition, their functional properties. V. Purification of Lymphocyte Subclasses

A. AFFINITY CHROMATOGRAPHY ON COLUMNS Affinity chromatography has been especially useful for the separation of large numbers of cells and provides an initial fractionation procedure for subsequent isolation techniques and functional studies. Most of the techniques reported have relied on the use of solid-phase supports to which are bound, either by adherence or by covalent linkage, a variety of molecules that have affinity for cell surface receptors, antigens, or other determinants. The solid supports most commonly used have included cross-linked dextrans (Sephadex G-200) (Schlossman and Hudson, 1973; Chess et al., 1974a), glass and acrylamide plastic beads (Degalon) (Wigzell and Anderson, 1969; Jondal et al., 1972). A primary factor in the choice of the solid support is the nonspecific retention of the cells. For example, it is known that B cells and monocytes will nonspecifically adhere to glass or nylon. Moreover, certain B and T cells will nonspecifically absorb to Degalon. For this reason we have chosen in our laboratory to use Sephadex G-200, which is a near-perfect filter for human lymphocytes and

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monocytes when specific antibodies are not covalently linked. During the last few years we have had experience with Sephadex G-200 covalently linked to antihuman F(ab)2as a cellular immunoabsorbent for the primary isolation and separation of surface Ig+ and Ig- lymphocytes. As shown in earlier studies, human B cells bind to Sephadex anti-F(ab), as a consequence of either intrinsic or cell surface Ig, whereas non-Ig-bearing cells (T plus null cells) do not bind. In addition, unlike anti-F(ab)2columns made on Degalon supports, Sephadex G-200 anti-F(ab)2allows most Ig- Fc receptor-bearing cells to pass through. One of the other major advantages of these columns is that the bound surface Ig-bearing cells can be recovered by competitive elution with human Ig. Quantitatively both the passed and recovered cells account for greater than 90% of the starting population of lymphocytes. Cells passing directly through the anti-F(ab)2columns contain less than 2% Ig-bearing lymphocytes, whereas those eluted from the column contain greater than 98% Ig+ cells and less than 2% E-rosetting cells. The passed population is heterogeneous with respect to E rosetting, since only 7 0 4 0 % of this population form E rosettes. Of importance is the fact that the separated populations remain functionally intact, can be recovered with minimal loss, and maintain their surface properties in vitro. In addition, the Sephadex anti-F(ab)2column techniques can be modified to specifically isolate and deplete cells bearing unique cell surface antigens which are recognized by specific antisera (Chess et al., 1976). Thus, Ig- cells can first be coated with rabbit anti-p23,30 and then passed over a Sephadex G-200 goat antirabbit Ig column. Cells passing through such columns can be shown to be p23,30. By competitive elution with rabbit y-globulin, the bound cells can b e recovered and shown to be p23,30+. Similarly, anti-TH1coated Ig- cells can be passed over similar columns and both THI+ and TH1- subclasses recovered. This general approach can obviously be utilized for any heteroantisera that recognize distinct differentiation antigens or other determinants on lymphocyte surfaces.

B. FLUORESCENCE-ACTIVATEDCELL SORTING The reader is referred to publications by Hertzenberg and his colleagues (Bonner et d.,1972; Loken and Herzenberg, 1975)describing detailed methodology and analysis of cell separation capabilities of the Becton-Dickinson fluorescence-activated cell sorter (FACS). This instrument analyzes and separates cells on the basis of either cell surface fluorescence or size and can provide a histogram of the number of positive fluorescent cells obtained against the intensity of fluores-

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cence. Individual subclasses of cells as defhed by either their scatter of fluorescence profile can be charged and specifically deflected and recovered for subsequent studies. Both direct and indirect fluorescent techniques can be used on the cell sorter. Utilizing the direct approach, cells are stained by resuspending approximately 2 to 3 x loe cells in 0.2 ml of fluoresceinated antibodies and incubated for 1 hour at 4°C. After 3 washes, the labeled cells can be processed on the FACS at approximately 500-1000 cells per second, and the intensity of fluorescence is recorded for each individual cell on the pulse height analyzer. Background fluorescence is determined by analyzing appropriate negative controls including cells labeled with fluoresceinated normal serum. Cell-sorter analysis of purified populations of cells isolated by immunoabsorbent chromatography has been particularly useful. For example, 98%of human B cells isolated by Sephadex anti-F(ab)zimmunoabsorbent columns are surface Ig+ as detected on the cell sorter. In contrast, less than 1% of the isolated T-cell populations bound surface Ig. It was also shown that the null-cell population was heterogeneous with respect to the quantity of surface Ig and that a small subclass of cells had a relatively low degree of fluorescence as detected on the FACS. This small population of cells, when analyzed by either direct or indirect techniques utilizing the same polyvalent anti-F(ab), reagents with fluorescence microscopy was not detectable as surface Ig+. Perhaps of more importance, with fluoresceinated rabbit antip23,30 sera it was shown that greater than 90% of B cells isolated by immunoabsorbent columns were intensely stained whereas less than 2% of the T cells were reactive (Schlossman et al., 1976). A subset of cells within the null-cell population accounting for approximately 2 0 4 0 % of the population is p23,30+. These data were important with respect to the analysis of the purity of the populations isolated b y column chromatography since the method used for detection of cells was distinct from the antibody used for primary isolation procedures. In more recent studies, purified T-cell populations were analyzed using indirect fluorescence on the cell sorter with the rabbit anti-THl antibody (Evans et al., 1977). The developing reagent was a horesceinated pepsin digested goat antirabbit F(ab)z. Anti-T", binding to B cells was not significantly different from that obtained with normal rabbit sera. The distribution of fluorescence binding to T cells with anti-TH1sera was Gaussian. Thus, greater than 90% of peripheral T cells reacted with anti-TH1sera, contrasting with a restricted number (5040%)of cells lysed by the same antisera using antibody and complement. It was therefore important to isolate low density-staining cells from high density-staining cells to ascertain whether cells bind-

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ing few antibody molecules correspond to TH1- subclasses as detected

by complement-mediated lysis. Thus, both low-density (lowest 25%) and high density binding (highest 25%) T cells were collected and analyzed by complement-dependent lytic assays, and it was shown that the TH1- subclass, i.e., the low density-staining cells were not lysed by treatment with anti-TH1+complement, whereas the high density-straining cells were lysed. As will be described below, these two subclasses were then analyzed for functional properties and shown to be distinct. C. ROSETTE DEPLETIONTECHNIQUES

The simple E- and EAC-rosetting techniques adapt themselves to widespread use as both analytic and cell separation tools (Mendes et al., 1973; Greaves and Brown, 1974; Wahl et d., 1974; MacDermott et aZ., 1975). The problems involved in utilizing these techniques for separation are similar to those encountered in analytical rosetting assays. In addition, the volume of lymphoid cells that can be handled is limited, since repeated rosetting is required before highly purified populations are obtained. With repeated rosettings, there are losses of cells, and the recovered cells may be contaminated with cells that influence the results obtained in functional assays. Further procedures directed at the removal of sheep red blood cells or EAC cells bound to lymphocytes in the rosetted population, such as lysis with ammonium chloride, may alter the functional capacity of the recovered lymphocytes. Nevertheless, rosetting techniques are particularly useful as a second step in the isolation of subclasses of cells. Thus, E- and EACrosette depletion techniques have been used to subfractionate the Igpopulation obtained from anti-F(ab), columns or on the FACS into E rosette-positive and E rosette-negative subclasses. After E depletion of Ig- cells, the resulting population is predominantly Ig- and E rosette negative. This population, which has been termed “null cells” is still heterogeneous with respect to other cell surface markers, including the p23,30 antigen, the Fc receptor, and the complement receptor. Functional properties of the p23,30+ Ig- E rosette-negative null-cell subclass will be described below. In addition, EAC depletion of the Ig- population of cells can be used to great advantage. The population remaining at the interface of Ficoll-Hypaque under these conditions comprises a class of cells which is greater than 92% E rosette positive and represents a highly purified population of T cells. T cells isolated by these methods are Ig-, E rosette positive, and EAC rosette negative and contain less than 1% p23,30+ cells. Further, only 5040% of these cells are lysed by the anti-TH1sera.

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D. OTHER SEPARATION TECHNIQUES Several other techniques are available for the separation of complex mixtures of viable cells into their component subsets. These techniques rely on biophysical differences that exist in various lymphocyte subclasses including plasma membrane electrical charge differences, density, and cell size. Thus, a number of methods have been developed utilizing cellular electrophoresis (Ambrose, 1965; Hayry et al., 1975), sedimentation over a variety of density gradients (Shortman et al., 1975; Raidt et al., 1968; Geha and Merler, 1974b), and velocity sedimentation (Miller, 1976) for separation of different cell populations. The reader is referred elsewhere for the details involved in these techniques. As a general rule, there remains considerable overlap in the functional and cell surface characteristics of cells isolated by these techniques, and it appears that their greatest usefulness will be in the definition of the heterogeneity of cells initially fractionated b y other methods. VI. The Functional Anolyrir of lroloted Humon Lymphocyte Subpopulotionr

A. GENERALCONSIDERATIONS The functional properties of isolated T, B, and null cells have been analyzed extensively with respect to a number of in vitro assays of

cell-mediated immunity. The data from our laboratory are summarized in Table I1 and point out the functional heterogeneity of the cells isolated by the methods described above. It is clear that a number of the assays of cell-mediated immunity in man discriminate functionally unique subpopulations of cells. Unique properties of T cells include their capacity to proliferate in response to specific soluble antigens, to recognize foreign HLA-D determinants in mixed lymphocyte culture (MLC), to recognize HLA-A and B determinants as the effector cells in cell-mediated lympholysis (CML), and to secrete some lymphokines, such as lymphocyte mitogenic factor (LMF). The only distinct functional property of B lymphocytes is their capacity to secrete Ig. However, it should be noted that B cells, in addition, are the only cells that have receptors for Epstein-Barr (EB) virus and allow EB viral replication. The characteristics of null cells that distinguish them from B cells are their capacity to function as the effector cell in antibody-dependent cellular cytotoxicity, and, perhaps equally interesting, their capacity to differentiate into granulocyte-forming colonies and erythrocyte-forming colonies. The latter two functions reflect the heterogeneity of the null-cell population. In contrast to the assays that appear to distinguish T, B, or null cells, a number of assays of cell-mediated immunity (Table 11) are subserved b y more than one

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HUMAN LYMPHOCYTE SUBPOPULATIONS TABLE I1 1MMUNOLOGIC FUNCTIONS O F HUMANLYMPHOCYTE SUBSETS in Vitro Immune functions

Pro1i ferative responses 1. Soluble antigen-triggered proliferation 2. Response to alloantigens in mixed lymphocyte culture MLC 3. Stimulating capacity in MLC Mediator production 1. Migration inhibitory factor (MIF) 2. Leukocyte inhibitory factor (LIF) 3. Lymphocyte mitogenic factor (LMF) Cytotoxic responses 1. Cell-mediated lympholysis (CML) of allogeneic cells 2. Antibody-dependent cellular cytotoxicity (ADCC) 3. Mitogen-induced nonspecific cytotoxicity Antibody production 1. Capacity for Ig synthesis in cell culture 2. Plaque-forming cells Miscellaneous functions 1. Precursors of granulocyte- and erythrocyte-forming cells, B cells, and T cells 2. Proliferative response to Epstein-Ban virus "

T

B

Null

++ + +

++

+ + +/-

+ + + + +

-

NT' NT -

+

+

+ + +

NT

+ +

NT. not tested.

subclass of cells. For example, migration inhibitory factor (MIF) is made b y both T and B cells, and it appears that B cells quantitatively account for most of the MIF produced by unseparated populations. Whether small numbers of T cells are required for B-cell MIF production remains controversial. Another mediator of cell-mediated immunity, leukocyte inhibitory factor (LIF), also appears to be produced by both T and B subpopulations, whereas LMF is produced exclusively by T cells. With respect to null cells, it is important to point out that a subclass of these cells can differentiate into Ig-secreting cells in uitro. This subclass bears the Ia-like determinant, p23,30, and thus may represent a subpopulation of B cells at a different stage in differentiation. In a subsequent section of this review we will focus attention on studies pertaining to the functional properties of subpopulations of lymphocytes and describe further the heterogeneity that exists within isolated T, B, and null-cell populations. IN RESPONSE TO SOLUBLEAND CELLULAR B. PROLIFERATION

ANTIGENS

It is known that purified T cells but not B cells from individuals previously sensitized to relatively complex antigens [purified protein

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L. CHESS AND S. F. SCHLOSSMAN

derivative (PPD), mumps, tetanus toxoid, etc.] respond by proliferation and incorporation of tritiated thymidine to specific soluble antigens i n vitro (Geha et al., 1973; Chess et al., 1974a,b). Using the ability to subfractionate T cells into TH1+ and T H I - subclasses, evidence has been obtained that the population of cells proliferating to specific soluble antigens are THI-,while those proliferating in response to alloantigens in MLC are TH1+ (Evans et d.,1977; Chess and Schlossman, 1977). For example, purified T cells from individuals sensitized to tetanus toxoid, PPD, and mumps antigen were treated in the presence of complement with media alone, normal rabbit serum, or anti-TH, sera. After treatment, T cells were washed extensively and cultured either in the presence or in the absence of soluble antigen and pulsed with ['Hlthymidine after 6 days. Although anti-THlsera lysed 60% of the cells, it had no significant effect on the antigen-induced proliferative response. More important, when identical cultures were cocultivated with mitomycin C-treated allogeneic cells in the MLC assay, the anti-TH1,but not the normal rabbit serum, eliminated the proliferative response. These data suggested that the THI+ cell, but not the THIcell, contained the MLC responsive cells, whereas the TH1-cells were capable of responding to specific soluble antigens. To determine whether T H 1 + cells could also respond to soluble antigens, the T H 1 + and THI- cells were isolated as described above on the FACS. The cells binding anti-THlwere divided into weak-binding (lowest 25%) or strong-binding (highest 25%) cells. It was shown that the lowestbinding cells (THI-by complement-mediated lysis) developed an excellent proliferative response to soluble antigens but did not react appreciably to allogeneic cells. In contrast, the isolated T H I + subclass were MLC responsive but did not proliferate in response to soluble antigens. These data support the view that the TH1 differentiation antigen was dissecting two unique subclasses of T cells, each subserving different functions; one programmed to respond in MLC, the other triggered by specific soluble antigens.

c. MEDIATOR PRODUCTION

BY

SUBCLASSES O F HUMAN

LYMPHOCYTES

The distinct cell surface determinants have permitted a reevaluation of mediator production by individual lymphocyte subclasses. Lymphocytes from individuals exhibiting delayed hypersensitivity are triggered in vitro by antigen to produce factors (lymphokines) with distinct biological properties including MIF (David, 1966; Bloom and Bennett, 1966; Rocklin et aZ., 1970),LMF (Maini et al., 1969),and LIF

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(Rocklin, 1974). Since the production of these factors correlated with the delayed skin reaction, it was generally assumed that the antigeninduced stimulation of these factors was mediated b y T cells. The development of cellular separation techniques has allowed for hrther dissection of the cell populations which interact and produce these factors. In studies using Ig- and Ig+ populations, it was shown that MIF was produced both by T and B cells (Rocklin et al., 1974). Further, it was shown that MIF produced b y B cells was indistinguishable from that produced by T cells and that B cells, despite their failure to proliferate in response to antigen, produced quantitatively more M I F than T cells. In these earlier studies, the possibility remained that small numbers of contaminating T cells could be contributing to the production of MIF or are themselves responsible for its production. This question was of particular importance, since many B cell functions are T-dependent. Moreover, in the guinea pig, MIF production by B cells appears to be T-cell dependent (Wahl and Rosenstreich, 1976). To further explore the cell populations involved in mediator production, cells were analyzed after treatment with anti-TH1sera or p23,30 sera (Evans et al., 1977; Chess and Rocklin, 1977). T cells from a number of individuals previously shown to exhibit hypersensitivity to either streptokinase-streptodornase (SKSD), PPD, or Candida antigens were treated with either normal rabbit serum (NRS) or anti-TH1in the presence of complement. The remaining T-cell population ( T H I - ) were tested for their capacity to elaborate MIF after specific triggering b y antigen. Treatment of T cells with NRS and complement had no significant effect on MIF production. In contrast, anti-TH1and complement abrogated the production of MIF, indicating that the MIF was produced b y the T H I + cells. It should be emphasized that the same T H 1 + subset ofcells did not proliferate in response to soluble antigens. To investigate the possibility that the TH1 + cells were contaminating isolated B-cell populations, B cells were treated with either NRS or anti-TH1and complement prior to triggering with antigen and assaying for MIF production. Neither treatment had any effect on B-cell production of MIF. These results demonstrate that the T-cell subclasses primarily responsible for MIF production play no demonstrable role in the production of MIF by B cells. However, these studies d o not formally exclude the possibility that contaminating T H i - cells, which by themselves do not produce MIF, may influence the production of MIF by B cells. In similar studies, treatment of unfractionated populations of lymphocytes with anti-TH1and complement did not affect MIF secretion. This finding is in accord with previous studies showing that B cells

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L. CHESS AND S . F. SCHLOSSMAN

quantitatively produce more MIF than T cells. Moreover, treatment of unfractionated populations of cells with anti-p23,30 and complement significantly reduced the MIF production but had no effect on the production of MIF by purified T cells. The data presented on MIF can be viewed with respect to clinical observations that lymphoid populations from some patients with immunodeficiency states contain cells that undergo antigen-induced proliferation but do not make MIF (i.e., patients with sarcoidosis and chronic mucocutaneous candidiasis) (Rocklin et al., 1971) whereas cells from other patients are capable of MIF production but do not proliferate (Levin et al., 1970; Kirpatrick et al., 1972; Whitcomb and Rocklin, 1973), i.e., patients with miliary tuberculosis, Wiskott-Aldrich syndrome, and candidiasis treated with transfer factor). We would suggest that patients whose cells are capable of being triggered by antigens to elaborate MIF but not to proliferate have functionally intact T H 1 + cells or B cells and an absence or functional impairment of the THl- subclass. Similarly, patients whose lymphocytes proliferate but do not secrete mediators may have intact TH1- cells and have impaired function at least with respect to mediator production in both TH1+ cells and B cells. In these patients it is entirely possible, and even likely, that other functions of THl+cells and B cells may remain normal, since mediator-producing cells may still represent subsets of THl+ cells and B cells. Lymphocyte mitogenic factor (LMF), unlike MIF, has been shown to be produced exclusively by T cells (Geha et aZ., 1973; Rocklin et aZ., 1974; Breard et al., 1977). To determine whether the same subclass of T cells produces LMF as makes MIF, populations of T cells were treated with anti-TH1plus complement and cultured with antigen for 48 hours, and LMF production was measured. Lysis of THl+bearing cells did not abolish the proliferative response of these cells to a variety of antigens; nevertheless, the supernatants from these cultures did not contain LMF activity (Evans et aZ., 1977). In contrast, treatment with normal rabbit serum plus complement had no effect. These results support the view that TH1+ cells produced both LM F and MIF. D. CELL-MEDIATED LYMPHOLYSIS The allograft reaction involves a complex sequence of cellular immune events that have been studied in detail using a variety ofin uitro model systems. These studies have demonstrated that one critical pathway in allograft rejection is the sensitization and subsequent differentiation of T cells to cytotoxic cells which recognize cell surface determinants genetically controlled by the major histocompatibility complex (Cerrottini and Brunner, 1974). A number of investigators

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have demonstrated during in vitro sensitization in one-way mixed leukocyte cultures (MLC) that effector T cells are generated which can specifically kill target cells bearing alloantigens in common with the sensitizing cell (Hayry and Defendi, 1970; Solliday and Bach, 1970; Lightbody et al., 1971). This phenomenon has been termed cell-mediated lympholysis (CML). Several aspects of the allogeneic (ML response are important for an understanding of the mechanism by which T cells kill either autologous or syngeneic tumors. The generation of cytotoxic cells requires a proliferative response which can be blocked b y antimitotic agents (Bach et al., 1972; Cantor and Jandinski, 1974).The majority of proliferating cells appear to recognize HLA-D determinants on the stimulating cell. Evidence for the requirement of interacting T cells has been suggested from studies which have shown that HLA-D reactive cells do not by themselves mediate killing, but instead provide helper interactions with a differentiating cytotoxic cell (Eijsvoogel et al., 1973; Schendel et al., 1973). Moreover, the cytotoxic cell recognizes serologically defined determinants (HLA-A and HLA-B) that are genetically distinct from the HLA-D locus. In the mouse, CML effector cells and proliferative helper cells are T lymphocytes and represent a functionally distinct subclass of Ly23 and Lyl cells, respectively (Cantor and Boyse, 1975a). In man, only the THI+subset of cells proliferates in response to alloantigens; anti-Ia sera, including anti-p23,30 and B cell alloantisera, are potent inhibitors of the MLC response (Wernet et al., 1975; Humphreys et al., 1976). Analysis of the T cells that differentiate into killer cells was performed using anti-TH1 sera. Treatment of killer cell populations generated after sensitization with anti-TH1antisera substantially, but not completely, reduces CML activity, suggesting that both MLC responsive and killer cells are T,, +. Further experiments will be needed to completely resolve this point.' Alloactivated T cells are not only effector cells in CML, but develop the capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). Lysis of alloactivated T cells with anti-p23,30 plus complement eliminated this function. Additional insights into the heterogeneity of T cells will be defined when other functions of activated T cells are investigated.

E. CELL-MEDIATED DESTRUCTION OF SYNCENEIC TUMOR CELLS Recently, in murine systems the principles of CML have been applied to the study of mechanisms involved in the generation of cytotoxic cells with specificity directed toward syngeneic cells that

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have been virally altered, chemically altered, or associated with tumors. Both the recognition and specificity of the effector phase of syngeneic killing appear to be dependent on either alteration of cell surface determinants controlled within the MHC or, alternatively, on antigens recognized in association with MHC determinants (Zinkernagel and Doherty, 1975; Gardner et al., 1975; Shearer et al., 1975; Schrader and Edelman, 1976). These examples of murine cytotoxicity against altered H-2 identical tissue have proved to be useful in the study of both tumor immunity and autoimmunity. In recent studies some mechanisms involved in the destruction of human acute leukemic cells by MHC-identical lymphocytes were examined (Sondel et al., 1976). Activation of human cytotoxic T lymphocytes (CTL) directed against serologically defined (SD) HLA-A and B determinants requires an active collaborative response to Ia surface molecules. A similar collaborative response may be important in the generation of cytotoxic cells to weak antigens in uitro. Thus, the addition of an allogeneic LD stimulus might provide the required helper signal needed to induce the cell-mediated killing of MHC-identical leukemic cells. These studies demonstrated that human lymphocytes in the presence of a third-party signal could be sensitized in uitro to recognize and destroy fresh leukemic blasts obtained from MHC identical siblings while not mediating destruction of normal lymphocytes or normal PHA blasts from other healthy MHC identical siblings. The antigens recognized on the leukemic cells should not be considered tumor-specific until further analysis. However, the MHC identity between the patients and siblings studied in these experiments demonstrated that the cytotoxic recognition of leukemic blasts was not a consequence of recognition of genetically controlled foreign MHC antigens. In addition, previous studies have demonstrated that healthy MHC identical siblings cannot generate CML against presumptive minor loci (non-MHC antigens) on each other’s lymphocytes even when sensitized in uitro in the presence of an unrelated third party (Sondel and Bach, 1976). Thus, the antigens recognized on the leukemic blasts are not conventional histocompatibility antigens (either MHC- or minor locus-controlled) that would be recognized as foreign on PHA blasts derived from normal lymphocytes. Whether these antigens are expressed on embryonic or other differentiated tissues remains open for further investigation. This question may be approached experimentally by using recently developed antigenspecific CML blocking techniques (Sondel and Bach, 1976). Certain aspects of these studies in relation to the cellular mechanism of in uitro sensitization and killing of MHC identical human

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leukemic cells are important. As noted above, murine studies by several groups have demonstrated that chemically or virally altered syngeneic cells can sensitize lymphocytes to become specifically cytotoxic to these altered cells. The antigenic alteration has been shown to specifically involve products of the MHC. In uitro destruction of H-2 identical tumor cells can also be directed at altered H-2 antigens. Thus, if spontaneously arising human leukemias were induced by interaction with a putative antigen-altered oncogenic virus, one would have predicted that the CML response to human leukemias would parallel those reported in murine systems. However, it was found that human leukemic cells alone rarely induce detectable antileukemic CML. The generation of antileukemic CML was enhanced by the addition of MHC-unrelated stimulating cells. In the murine system chemically or virally altered syngeneic cells alone are sufficient to stimulate CML; whether the addition of an allogeneic trigger would augment the cytotoxic response has not yet been determined. The necessity for and role of the third-party stimulation in augmenting sensitization to human leukemic blasts remains to be clarified. Allogeneic stimulating cells may provide the required proliferative helper cell stimulus analogous to that observed in the generation of CTLs to alloantigens. Alternatively, allogeneic lymphocytes may b e necessary to overcome the action of suppressor T cells. In more recent studies of the killing of syngeneic leukemic blasts by siblings’ lymphocytes, we have demonstrated that the effector cell in this system is a T lymphocyte; studies are now under way to see whether the subclass of cells required for the generation of these killer cells is similar to the ones generated against allogeneic target cells.

HUMAN PERIPHERALBLOOD B-CELL FUNCTION The one unequivocal function of B cells is their capacity to synthesize and secrete immunoglobulins. It is clear from studies in a number of mammalian species that clones of antibody-forming precursor cells are genetically programmed prior to interaction with antigens to synthesize antibody molecules of highly restricted specificity. Mechanisms by which these clones are triggered to differentiate into antibody-forming cells depend on the maturational state of the B cells, on their location within a variety of lymphoid compartments and, perhaps equally important, on the nature of signals delivered to the cell surface. Thus, although some antigens appear to be capable of triggering precursor B cells alone (“T independent”) the majority require additional signals that in many instances can b e shown to origiF.

ANALYSIS OF

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L. CHESS AND S . F. SCHLOSSMAN

nate from antigen-triggered T cells. The nature of the specific T cellgenerated signals is poorly understood at present. Some can be nonspecifically bypassed by: (1) macrophages and their products (Calderon et al., 1975); (2) nonspecifically activated T cells (Mond et al., 1972; Rubin and Coons, 1972; Amerding and Katz, 1974); and (3) polyclonal activators, such as lipopolysaccharide (LPS) or pokeweed mitogen (PWM) (Andersson et al., 1972; Parkhouse et al., 1972; Coutinho and Moller, 1975). The relationship between nonspecific and antigen-specific T-cell helper effects is critical to our understanding of human B-cell functions. Most studies investigating human B-cell Ig synthesis have utilized PWM, since human cells respond poorly if at all to LPS or soluble anti-Ig. In a number of studies, human B cells have been found to respond to PWM by blast transformation, thymidine uptake, plasma cell development and Ig secretion. Optimal differentiation of B cells can be shown to be dependent on the presence of either stimulated T cells, macrophages, or their products. Although these studies have provided important information concerning B cell function and have allowed the analysis of B cell immunodeficiency disorders, defining the mechanisms of human B cell activation has been limited by the difficulty in developing specific in vitro systems. In particular, plaque-forming assays have facilitated our understanding of the mechanisms and nature of signals required for specific antibody production in other species. Several investigators have recently reported the primary in vitro induction of antibody synthesis to hapten-conjugated T-dependent carriers or sheep erythrocytes (Watanabe et al., 1974; Dosch and Gelfand, 1976; Fauci et al., 1976). The methods used so far lack the ease and reproducibility observed in murine systems. The difficulties encountered may reflect in part the fact that human peripheral B cells may be less differentiated and more easily tolerized than splenic and lymph node cells. Human peripheral blood B lymphocytes triggered nonspecifically by polyclonal mitogens to secrete antierythrocyte antibodies in vitro continue to bear Ia determinants (Friedman et al., 1977).Since it is known that Ia determinants are not readily detected on plasma cells, it has been suggested that peripheral blood lymphocytes may be more immature than B lymphocytes found in other lymphoid compartments (Schlossman et al., 1976). In this regard we and others have shown that B cells are capable of producing a significant plaque-forming response to sheep erythrocytes when triggered by PWM in the absence of antigen. In contrast, unfractionated populations and purified populations of T cells are not.

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The PWM activated plaque-forming response represents true antibody synthesis since: (1) it can be inhibited by rabbit antihuman Ig; (2) it requires complement to lyse the plaques; (3) each plaque contains at least one, and up to ten central lymphoid cells; and (4) treatment with cycloheximide at doses known to inhibit protein synthesis inhibits plaque formation (Friedman et al., 1977). These controls are essential since many artifacts arise in human B-cell plaque-forming systems to heterologous erythrocytes. A more “physiologic” trigger for B cells is the soluble products of activated T cells. Supernatants from antigen-triggered T cells containing lymphokines can induce B cells to differentiate into antibodysynthesizing cells. A requirement for optimal triggering of B-cell plaque formation is prior binding and elution from anti-F(ab)2columns. B cells recovered from nylon wool columns or b y other techniques are not as readily triggered by either PWM or activated T-cell supernatants. These studies suggest that partial activation of B cells by antiF(ab), columns may provide an initial signal and stimulate antigen triggering or allow for subsequent triggering by PWM or T-cell products. Further, the addition of sheep erythrocytes to PWM-triggered B cells does not enhance the number of plaque-forming cells, but instead suppresses the response. The suppression is specific, since the response to unrelated erythrocytes is not diminished (Friedman and Chess, 1977). These data support the notion that peripheral blood B cells may be easily tolerized and, together with the observation that antibody-secreting B cells retain Ia antigens, provide support for the view that circulating human B cells are immature. The pokeweed-induced plaque-forming system is less ideal than testing with antigen-specific T cell-dependent plaques. Nevertheless, these systems have allowed investigation of a number of the features of B-cell differentiation in man. We have studied the effects of the anti-p23,30 sera on plaque-forming cells following stimulation with PWM and T cell supernatants. The following observations were made: (1) anti-p23,30 but not normal rabbit serum markedly reduces the generation of PFCs, but only partially suppresses the proliferative response to PWM; (2) the induction of both PFC and B cell proliferation b y soluble products of activated T cells is markedly inhibited b y anti-p23,30; (3) the inhibitory effect of anti-p23,30 in the mitogeninduced PFC response can be demonstrated only when the antiserum is present in the early phases of cell culture-its effects are minimal at later stages; and (4) the p23,30 antigen is retained by the more “differentiated” antibody-secreting cells after 6 days of culture (Friedman et al., 1977). These results suggest that the Ia-like determinant, p23,30,

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L. CHESS AND S. F. SCHLOSSMAN

although present on mature PFCs, plays its major role during the early differentiation of activated precursor B cells into antibody-forming cells. G. CHARACTERIZATION AND ISOLATION OF REGULATOR CELLS I N HUMAN PERIPHERAL BLOOD It is apparent in murine systems that both the type and intensity of the cellular and humoral immune responses to antigen can be homeostatically regulated b y a complex series of interactions involving distinct classes of lymphocytes and macrophages. Distinct subclasses of T cells are capable of helper, amplifier, and suppressor effects on the development of T cell-mediated functions such as cytotoxicity and B-cell secretion of Ig (Gershon, 1974; Cantor and Boyse, 1976). Two interesting aspects of these complex cellular interactions are (1) that the regulatory influences of T-cell subclasses (both helper and suppressor) are under relatively strict genetic control by genes linked to the MHC (reviewed by Katz and Benacerraf, 1975) and (2) that a number of regulatory functions of these subclasses are mediated by factors that influence other T-cell subclasses, B cells, or macrophages (Tada et al., 1976; Taussig and Munro, 1974; Rich and Pierce, 1974). In man, it is now clear that regulatory cells exist and for the most part are found in the Ig- subclass of cells and within adherent cell populations (Waldmann et al., 1974; Siegal et al., 1976; Friedman et al., 1977). For example, it has been shown that the formation of PWMinduced B-cell plaques can be suppressed by the addition of autologous Ig- cells. Furthermore, this suppression can be augmented by prior addition of concanavalin A (Con A). Treatment of Con A-activated Ig- cells with anti-TH1serum plus complement, while eliminating approximately 50% of the cells, had no effect on the suppressor cell function. Thus, one subclass of human suppressor cells may exist within the T H I - subclass. In addition, other subpopulations of human peripheral mononuclear cells can also suppress the differentiation of B cells triggered by polyclonal mitogens. Adherent cells within the Ig- population, e.g., like the nonadherent population, can also suppress B cells. Whether adherent Ig- cells are identical to nonadherent suppressor T cells remains to be determined. In addition, evidence has been presented by a number of investigators using purified human T cells that at least one subclass of T cells has the capacity to augment B-cell differentiation into Igsynthesizing cells. These putative helper T-cell subclasses have been recognized both by heteroantisera directed at unique subclasses of T cells and by alloantisera present in patients with juvenile rheumatoid arthritis and are directed against specificities of subclasses of T cells

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that suppress B-cell secretion of Ig (Strelkauskas et al., 1977). Moreover, anti-TH,antibody eliminates that subclass of T cells which secrete factors capable of augmenting B-cell differentiation (Breard et al., 1977). Thus, the concept is emerging that in man there are distinct subclasses of T cells that can initiate, help, and suppress the differentiation of B cells. Clarification of the specificity of these effects will have to await more precise antigen-specific B cell functional assays. HETEROGENEITY OF NULL CELLS H. THE FUNCTIONAL As indicated initially, considerable evidence exists that there are subclasses of lymphocytes which lack the precise surface properties of the predominant differentiated classes of T and B lymphocytes. In man, this null-cell subclass is distinguished from T cells by its failure to form spontaneous rosettes with sheep erythrocytes and from B cells by its lack of detectable surface Ig. Further, the null cell is heterogeneous both with respect to the complement and Fc receptors, and, perhaps of greater importance, the null-cell subclass is heterogeneous with respect to the Ia determinant, p23,30. Approximately 2 0 4 0 % of the null cells isolated b y immunoabsorbant anti-F(ab)2chromatography followed by E-rosette depletion, are both complementreceptor positive and p23,30 positive (Chess et a/., 1976). Functional studies of the null-cell subclass have indicated that null cells are distinct from mature T cells (MacDermott et ul., 1975). Null cells do not proliferate in response to soluble or allogeneic cell surface antigens, whereas T cells do; and they do not kill allogeneic lymphocytes by direct CML. In addition, null cells, but not T cells, are the effector cells in ADCC. In contrast, the p23,30-positive subclass of null cells is functionally similar to B cells in that both subclasses spontaneously secrete Ig. However, the null-cell subclass, unlike circulating surface Ig+ B cells, effect ADCC. Treatment of null cells with anti-p23,30 plus complement depletes both their capacity to synthesize Ig in cell culture and their capacity to effect ADCC. The simplest interpretation of these findings is that the ADCC effector cell (i.e., the E-, Ig-, p23,30+, complement receptor-positive) in man and Igsynthesizing cell (E-, Ig+) bear close relationships and may represent different stages of B-cell differentiation. However, it is important to point out that the null cell bearing p23,30 is distinct from most peripheral B cells in being nonadherent, non-Ig bearing, and capable of eliciting the ADCC reaction. Whether there still exists a heterogeneity of cell types within the p23,30+, Ig-, EAC+ subclass that would distinguish the ADCC-reactive cell from the Igsynthesizing cell cannot be resolved with the available reagents. It is important to emphasize that approximately 70% of the null-cell

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subclass is p23,30- and the function of most of these cells remains largely unknown. Some of these cells have the capacity to differentiate, after appropriate triggering, in cell culture into granulocyte colonies (Richman and Chess, 1977),erythrocyte colonies (Nathan and Chess, 1977),and E + T cells. Thus, the null compartment of peripheral human mononuclear cells appears to contain precursor cells capable of generating the entire spectrum of hematopoietic and lymphopoietic cells. Whether all these precursors will be distinguished with antigens on their surface analogous to p23,30 remains to be determined.

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