Human b-cell differentiation. I. Analysis of immunoglobulin heavy chain switching using monoclonal anti-immunoglobulin M, G, and A antibodies and pokeweed mitogen-induced plasma cell differentiation.

Monoclonal antibodies were used to examine the immunoglobulin isotypes expressed by B lymphocyte precursors of IgM, IgG, IgA, and IgA2 plasma cells. Plasma-cell differentiation was induced by the addition of pokeweed mitogen to cultures of blood mononuclear cells. Anti-mu, -gamma, -alpha, and -alpha 1 antibodies were used in some experiments to inhibit differentiation of B lymphocytes bearing these heavy chain isotypes, and for selective removal of B lymphocyte precursors before culture with pokeweed mitogen in other experiments. Three major subpopulations of B lymphocyte precursors were identified: (a) a subpopulation of surface (s) IgM+ precursors of IgM plasma cells that did not express IgG or IgA isotypes, (b) a subpopulation of sIgG+ precursors of IgG plasma cells of which approximately one-half bore some IgM and none had detectable IgA receptors, and (c) a subpopulation of sIgA+ precursors of IgA plasma cells; one half of these precursors could be shown to express functional IgM receptors but none were found to express IgG receptors. The sIgA subpopulation could be further subdivided into sIgA1+ precursors of IgA1 plasma cells and IgA1-negative precursors of IgA2 plasma cells. These results suggest that normal human B cells can switch from mu directly to each of the other heavy chain isotypes, and that these represent the main switch pathways.

Human B cell differentiation. II. Pokeweed mitogen-responsive B cells belong to a surface immunoglobulin D-negative subpopulation.

Surface immunoglobulin D (IgD)-positive lymphocytes precoated with monoclonal anti-delta antibody were selectively removed from blood mononuclear cell preparations by "panning" and by fluorescence-activated cell sorter. The depletion of sIgD+ cells did not significantly affect plasma cell responses to pokeweed mitogen PWM). PWM-responsive B cells lacking sIgD and mouse erythrocyte receptors preferentially sedimented in lower density fractions of a discontinuous Percoll gradient, and sIgD-negative B cells were found to have a larger mean diameter than IgD-positive cells. We conclude that PWM-responsive B cells represent a distinct subpopulation of relatively large cells that have ceased to express receptors for mouse erythrocytes and surface IgD.

Impairment of T and B cell development by treatment with a type I interferon.

Type I interferons alpha and beta, naturally produced regulators of cell growth and differentiation, have been shown to inhibit IL-7-induced growth and survival of B cell precursors in vitro. After confirming an inhibitory effect on B lymphopoiesis in an ex vivo assay, we treated newborn mice with an active IFN-alpha2/alpha1 hybrid molecule to assess its potential for regulating B and T cell development in vivo. Bone marrow and splenic cellularity was greatly reduced in the IFN-alpha2/alpha1-treated mice, and B lineage cells were reduced by >80%. The bone marrow progenitor population of CD43+B220+HSA- cells was unaffected, but development of the CD19+ pro-B cells and their B lineage progeny was severely impaired. Correspondingly, IL-7-responsive cells in the bone marrow were virtually eliminated by the interferon treatment. Thymus cellularity was also reduced by >80% in the treated mice. Phenotypic analysis of the residual thymocytes indicated that the inhibitory effect was exerted during the pro-T cell stage in differentiation. In IFN-alpha/beta receptor-/- mice, T and B cell development were unaffected by the IFN-alpha2/alpha1 treatment. The data suggest that type I interferons can reversibly inhibit early T and B cell development by opposing the essential IL-7 response.

Postnatal expansion of the natural killer and keller cell population in humans identified by the monoclonal HNK-1 antibody.

Human natural killer (NK) and killer (K) cells were directly enumerated using a monoclonal antibody (HNK-1) and an immunofluorescence assay. The frequency of cells bearing surface HNK-1 antigen was very low in the newborn (less than 1.0%) and increased progressively through childhood and into adult life. This was correlated with an age-related increase in functional NK and K cell activities. Males had a slightly higher proportion of HNK-1+ cells than females. In addition to HNK-1 expression on the surface membrane, a prominent cytoplasmic expression of HNK-1 antigen was found in some but not all surface HNK-1+ cells. The cytoplasmic accumulation of HNK-1 molecules appeared to occur in more mature cells of this lineage.

Cellular distribution, regulation, and biochemical nature of an Fc alpha receptor in humans.

In these studies, we characterize an Fc receptor (FcR) for IgA that is present on human granulocytes, monocyte/macrophages, and their corresponding cell lines. Receptor expression appears to be constitutive but can be selectively upregulated on monocyte cell lines by stimulation with a phorbol ester and polymeric IgA. Both the induction requirements and ligand specificity of the IgA receptor differ from the IgG receptors, Fc gamma R I, II, and III, that are also expressed on monocytes and granulocytes. IgA binding to the cell surface receptor is mediated via the Fc alpha region. The Fc alpha R is a heterogenously charged, approximately 60-kD molecule with an isoelectric point of 4.5-5.6 that binds monomeric or polymeric IgA1 and IgA2 molecules. This transmembrane glycoprotein appears to be composed of 32- and 36-kD protein cores with multiple N-linked carbohydrate moieties. We conclude that this Fc alpha R represents a novel member of the FcR family that may have a distinctive role in host defense.

Thymic origin of embryonic intestinal gamma/delta T cells.

Current evidence suggests both thymic and extrathymic origins for T cells. Studies in mice favor an in situ origin for a prominent population of intestinal intraepithelial lymphocytes that express gamma/delta T cell receptor (TCR). This developmental issue is explored in an avian model in which the gamma/delta lymphocytes constitute a major T cell subpopulation that is accessible for study during the earliest stages of lymphocyte development. In the chick embryo, cells bearing the gamma/delta TCR appear first in the thymus where they reach peak levels on days 14-15 of embryogenesis, just 2 d before gamma/delta T cells appear in the intestine. Using two congenic chick strains, one of which expresses the ov antigen, we studied the origin and kinetics of intestinal colonization by gamma/delta T cells. The embryonic gamma/delta+ thymocytes homed to the intestine where they survived for months, whereas an embryonic gamma/delta- thymocyte population enriched in thymocyte precursors failed to give rise to intestinal gamma/delta+ T cells. Embryonic hemopoietic tissues, bone marrow, and spleen, were also ineffective sources for intestinal gamma/delta+ T cells. Intestinal colonization by gamma/delta+ thymocytes occurred in two discrete waves in embryos and newly hatched birds. The data indicate that intestinal gamma/delta T cells in the chicken are primarily thymic migrants that are relatively long-lived.

T cell hybrids that express a VH idiotope-related determinant on a glycoprotein distinct from H-2, Thy-1, and Lyt-1 molecules.

Two mouse monoclonal antibodies to chicken immunoglobulin VH-associated idiotypes (Id), CId-1 and CId-2, were used as probes for Id determinants on mouse T cells. CId-1, which recognized chicken antibodies to N-acetyl glucosamine (NAGA), and approximately 0.4% of chicken T lymphocytes also reacted with approximately 0.2% of BALB/c splenic Thy-1.2+ cells. When enriched CId-1+ splenic T cells from NAGA-immune BALB/c mice were fused with the AKR thymoma BW 5147 cell line, 2 of 72 resulting hybrids, termed CId-1A and CId-1B, were reactive by indirect immunofluorescence with the CId-1 antibody. CId-1 determinants were expressed both in the cytoplasm and on the cell surface. Immunofluorescence studies revealed that both CId-1+ T cell hybrids were phenotypically identical: CId-2-/Ig-/Lyt-1+2-/Thy-1.2+/II-2d+/I-Ad-/I-Ak-/I-Jd+/I-Jk+. Incubation of CId-1B hybrid cells with concanavalin A or lentil lectin resulted in capping of the CId-1 determinant, whereas incubation with pokeweed mitogen, lipopolysaccharide, phytohemagglutinin, and wheat germ agglutinin had no effect on the cell surface distribution of the CId-1 molecule. Trypsin or pronase treatment resulted in the loss of detectable CId-1 determinant on the cell surface. Treatment of CId-1B cells with tunicamycin also reduced the immunofluorescence intensity of the surface CId-1 determinant, but had no effect on its cytoplasmic expression. CId-1 antibody-induced capping of the CId-1 marker did not affect the surface distribution of Lyt-1, Thy-1.2, H-2d, I-Jd, or I-Jk molecules. Conversely, capping of I-Jd and I-Jk determinants did not alter the surface distribution of CId-1. These results suggest that the CId-1 determinant is on a glycoprotein that is not physically linked to the Lyt-1, Thy-1.2, H-2d, I-Jd, and I-Jk molecules. The clonal restriction of CId-1 expression by T cells suggests that the CId-1+ molecule could be a T cell antigen receptor.

Ontogeny of B cells in the chicken. I. Sequential development of clonal diversity in the bursa.

The initial development and distribution of lymphocytes expressing surface IgM (sIgM) and of specific antigen-binding cells (ABC) were studied in the chicken in an attempt to gain information on the process by which B-cell diversity is generated. The antigens used were sheep erythrocytes (SE), keyhole limpet hemocyanin (KLH), and poly-L(Tyr, Glu)poly-D,L-Ala-poly-L-Lys (TGAL). The results indicate that generation of the total sIgM-positive population begins in the bursa and that specific clones of ABC develop in a fixed sequential pattern which is not influenced by either deprivation of or deliberate exposure to exogenous antigens. Cells bearing sIgM by immunofluorescence (IgM-positive cells) were detected first in the bursa on the 12th day of incubation, KLH-ABC and TGAL-ABC by the 16th day, and SE-ABC on the 18th day. The doubling time of the sIgM-positive population of bursal cells was determined to be approximately 10 h before significant antigen-independent seeding to the spleen began a few days before hatching. Clonal expansion of SE-ABC in the bursa also appeared to be antigen independent as was the initial development of SE-ABC in the blood and spleen which ceased abruptly after bursectomy at hatching. Specific ABC were observed to develop in multiple bursal follicles as small foci of ABC among the much larger total population of sIgM-positive cells within an individual follicle. Intravenously infused SE-ABC homed to the embryonic spleen but not to the bursa. The results are interpreted as favoring a hypothetical model in which individual stem cells give rise to multiple clones of B cells by a predetermined pattern of sequential expression of variable region genes.

Fate of surrogate light chains in B lineage cells.

Biosynthesis of the immunoglobulin (Ig) receptor components and their assembly were examined in cell lines representative of early stages in human B lineage development. In pro-B cells, the nascent surrogate light chain proteins form a complex that transiently associates in the endoplasmic reticulum with a spectrum of unidentified proteins (40, 60, and 98 kD) and Bip, a heat shock protein family member. Lacking companion heavy chains, the surrogate light chains in pro-B cells do not associate with either the Ig(alpha) or Ig(beta) signal transduction units, undergo rapid degradation, and fail to reach the pro-B cell surface. In pre-B cells, by contrast, a significant portion of the surrogate light chain proteins associate with mu heavy chains, Ig(alpha), and Ig(beta) to form a stable receptor complex with a relatively long half-life. Early in this assembly process, Bip/GRP78, calnexin, GRP94, and a protein of approximately 17 kD differentially bind to the nascent mu heavy chains. The 17-kD intermediate is gradually replaced by the surrogate light chain protein complex, and the Ig(alpha) and Ig(beta) chains bind progressively to the mu heavy chains during the complex and relatively inefficient process of pre-B receptor assembly. The results suggest that, in humans, heavy chain association is essential for surrogate light chain survival and transport to the cell surface as an integral receptor component.

Biochemical nature of an Fc mu receptor on human B-lineage cells.

An IgM-binding protein of approximately 60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (Fc microR). The Fc microR can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation. Receptor expression is not directly linked with IgM production, as both mu- pre-B cells and isotype-switched B cells may express the Fc microR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with O-linked, but no N-linked, oligosaccharide. The Fc microR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fc microR is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens.

Immunoglobulin VH determinants defined by monoclonal antibodies.

Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

Ultrastructural studies of human lymphoid cells. mu and J chain expression as a function of B cell differentiation.

J chain expression was examined as a function of the stage in differentiation along the B cell axis in humans. Intracellular distribution of J and mu chains in leukemic HLA-DR+ null and pre-B cells, and in normal B cells stimulated with pokeweed mitogen (PWM) was determined by immunoelectron microscopy and radioimmunoassay (RIA). J chain was detected in leukemic null and pre-B cells on free and membrane-bound ribosomes in the cytoplasm, or on perinuclear cisternae. Mu chain was found on free ribosomes and ribosomal clusters in leukemic pre-B cells but was absent in the leukemic null cells. In pre-B cell lines, mu chain was seen within rough endoplasmic reticulum (RER) and the Golgi apparatus whereas J chain was not detected in these organelles. However, both mu and J chain were detected in RER and the Golgi apparatus of immature and mature plasma cells induced by PWM stimulation of normal peripheral blood lymphocytes. Low levels of J chain were also detected by RIA in lysates of leukemic null and pre-B cells. Most of the intracellular J chain became detectable after reduction and alkylation of cell lysates, and free J chain was not found in the culture supernatants. The amount of intracellular and secreted immunoglobulin-bound J chain increased dramatically after PWM stimulation of peripheral blood lymphocytes. The majority of J chain-positive cells seen over an 8 d culture interval were lymphocytes and lymphoblasts, while mu chain was found primarily in plasma cells. These results suggest that J chain expression precedes mu chain synthesis during B cell differentiation and that a combination of the two chains for secretion is not initiated until the onset of plasma cells maturation.

Suppression of immunoglobulin class synthesis in mice. I. Effects of treatment with antibody to -chain.

Germfree BALB/c mice have been treated from birth with intraperitoneal injections of purified goat antibodies to mouse IgM. The treated mice, and controls which had received an equivalent amount of goat gamma-globulin, were sacrificed at 8 or 13 wk of age. Compared to controls, mice given anti-micro (a) had very few germinal centers in spleen and lymph node, (b) had decreased numbers of mature plasma cells synthesizing IgM and IgG1 in spleen, and virtual absence of IgA-synthesizing plasma cells in the gut, (c) had greatly diminished numbers of B lymphocytes bearing membrane-bound immunoglobulins of the IgM, IgG1, IgG2, and IgA classes in spleen, (d) had reduced synthesis of IgM, IgG2, and IgA by in vitro spleen cultures, and (e) had significant decreases in serum levels of IgM, IgG1, IgG2, and IgA. The treated animals failed to make antibodies to ferritin after hyperimmunization, and lacked natural antibodies to sheep erythrocytes. These results indicate that cells ultimately committed to synthesis of IgG1, IgG2, and IgA immunoglobulins are derived from cells which have expressed IgM determinants at an earlier stage of differentiation. They are consistent with a proposed two-stage model for plasma cell differentiation. The first stage is antigen independent, involves sequential activation of Cmicro, Cgamma, and Calpha genes by progeny of a single stem cell, and results in the formation of B lymphocytes bearing membrane-bound recognition antibodies of each class. The second, antigen-dependent, stage results in formation of mature plasmacytes and memory cells.

A large subpopulation of avian T cells express a homologue of the mammalian T gamma/delta receptor.

This report describes an avian TCR molecule, TCR1, whose molecular characteristics, signal-transducing property, and tissue distribution suggest that it is a homologue of the mammalian TCR-gamma/delta. TCR1+ cells are the first to be generated in the thymus during ontogeny, preceding other T3+ cells by approximately 3 d. Unlike their mammalian counterpart, TCR1+ cells constitute a relatively large subpopulation of peripheral T cells in mature chickens. These results suggest a phylogenetically important role for this receptor in T cell development and function.

Identification of a T3/T cell receptor complex in chickens.

A mouse mAb, CT-3, recognizes on chicken T cells a complex of three polypeptides, Mr 20,000, 19,000, and 17,000, two of which are N-glycosylated. The CT-3 antibody is mitogenic for chicken T cells, and it coprecipitates two additional polypeptides of Mr 49,000 and 38,000 in lysates of T cell membranes. Ontogeny studies revealed that 5-6 d after thymic influx of hemopoietic stem cells, their thymocyte progeny begin to express the T3/TCR complex. After hatching 1 wk later, the CT-3+ cells begin splenic migration in large numbers.

Ethnic differences in the lymphocyte proliferative response induced by a murine IgG1 antibody, Leu-4, to the T3 molecule.

The mitogenic effects of isotypically diverse antibodies to the T3 molecule were examined in genetically diverse population groups. Whereas the OKT3 antibody (IgG2a) was mitogenic for blood mononuclear cells from all individuals tested, the 38.1 antibody (IgM) was consistently nonmitogenic. In contrast, studies of the mitogenic effects of the Leu-4 antibody (IgG1) revealed striking ethnic differences. More than 80% of Caucasians and Negroes were good Leu-4 responders, whereas most individuals of Asian origin, including Indian, Japanese, and Chinese, were either Leu-4 nonresponders or Leu-4 low responders. However, the majority of American Indians, as well as a significant minority of Chinese, were good responders. Cell separation studies confirmed that monocytes govern the different mitogenic effects of the anti-T3 antibodies. The results reveal interesting ethnic differences in monocyte accessory function probably mediated via the Fc-gamma receptor, in the stimulation of T lymphocytes by an IgG1 antibody against the T3 molecule.

Pre-B and B cells in rabbits. Ontogeny and allelic exclusion of kappa light chain genes.

Pre-B cells in developing rabbits were identified by immunofluorescence as cells containing small amounts of cytoplasmic IgM (cIgM) but lacking surface immunoglobulin (sIg). During ontogeny the first pre-B cells appeared in fetal liver at 23 days gestation, 2 days before the appearance of sIgM+ B lymphocytes. Pre-B cells were relatively frequent in fetal and adult bone marrow, but were not found in other tissues except rarely in fetal spleen. Allelic exclusion is apparently established at this early stage of development, because individual pre-B cells and B lymphocytes from heterozygous rabbits expressed only one of the alternative alleles in amounts sufficient for detection. Development of isotype diversity among rabbit B lymphocytes followed the general pattern seen in mouse and man. sIgM+ cells were detected before birth. Expression of sIgG was detected in neonatal rabbits on cells which were also sIgM+ but in older animals most sIgG+ cells lacked sIgM. Cells bearing sIgA were not found until 5-6 days of age, and had no other isotype on their surface.

Functional analysis of two human T-cell subpopulations: help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG.

Subpopulations of thymus-derived T lymphocytes bearing receptors for either IgM or IgG molecules were isolated from human peripheral blood. Those with receptors for IgM (T.M) provided help in a cell dose-dependent fashion for the pokeweed mitogen-induced differentiation of B lymphocytes in vitro, whereas cells with receptors for IgG (T.G) did not. T.G cells, on the hand, efficiently suppressed the differentiation and proliferation of B cells in the pokeweed system in the presence of helper T.M cells. This suppressive activity of T.G cells required prior interaction of the T.G cells with immune complexes. The helper activity of T.M cells was relatively radioresistant while the suppressor activity of T.G cells was radiosensitive. The results indicate that helper and suppressor functions of human T lymphocytes in this model system are mediated by different subpopulations of T cells which can be distinguished by their ability to bind IgM or IgG immune complexes, respectively.

The functions of the thymus system and the bursa system in the chicken.

The bursa of Fabricius and the thymus are "central lymphoid organs" in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Surgical removal of one or both of these organs in the newly hatched chicken, followed by sublethal X-irradiation the next day, has permitted recognition of two morphologically distinct cell systems in the "peripheral lymphoid tissues" of the spleen, gut, and other organs, and clear definition of the separate functions of each cell system. The thymus-dependent development is represented morphologically by the small lymphocytes of the circulation and the white pulp type of development in the tissues. As in mammals, the thymus-dependent tissues of the chicken are basic to the ontogenesis of cellular immunity: graft versus host reactions, responses of delayed hypersensitivity and homograft rejection; and play a less clearly defined role in the antibody response to at least some antigens. Thymectomized-irradiated chickens are deficient in all these responses, and grow more slowly than any of the other experimental groups. In these animals germinal centers, plasma cells, and capacity for immunoglobulin synthesis remain intact. The bursa-dependent development is represented morphologically by the larger lymphocytes of the germinal centers and the plasma cells, and functionally by the immunoglobulins. Bursectomized-irradiated chickens are agammaglobulinemic and unable to produce detectable antibody despite intense, repeated stimulation with bovine serum albumin and Brucella abortus organisms. The thymus-dependent development in these animals seems to be normal; they have adequate numbers of lymphocytes in the circulation and tissues, are able to reject skin homografts, though more slowly than usual, and to exercise graft versus host reactions. The short life span of these chickens has precluded adequate study of responses of delayed hypersensitivity. There was no evidence of significant impairment of reticuloendothelial function in either the bursectomized-irradiated or the thymectomized-irradiated group, as judged by the clearance of colloidal gold and I(131)-tagged keyhole limpet hemocyanin.

The IgA system. I. Studies of the transport and immunochemistry of IgA in the saliva.

1. Five patients with congenital or acquired agammaglobulinemia, lacking detectable IgA in serum or saliva, were transfused with 1 to 2 liters of normal plasma. In 2 of these patients IgA was demonstrated in parotid saliva collected after transfusion, but in none of the 5 was salivary IgG or IgM found. This observation indicates the selective transport of IgA into saliva. 2. The observation by others of an immunochemical difference between serum and sahvary IgA globulin was confirmed. In contrast to serum IgA, salivary IgA is attached to a protein having antigenicity which migrates as a gamma(1) globulin. We have termed this protein component "transport piece". 3. The transport piece has been found in an unbound form in the saliva of persons completely lacking IgA: agammaglobulinemic patients, ataxia-telangiectasia patients, a healthy person lacking IgA, and a newborn infant. Free transport piece still occurs in the normal child's saliva after IgA production begins. By adulthood there is usually no free transport piece in the saliva. 4. Heat-aggregated salivary IgA, like heat-aggregated serum IgA, does not fix complement. 5. Our findings offer support for the view that there is a distinct local antibody system for the protection of the mucous surfaces.