Differentiated B lymphocytes. Potential to express particular antibody variable and constant regions depends on site of lymphoid tissue and antigen load.

B cells have the potential to respond to an antigen by producing antibodies with a variety of variable and constant regions. We have quantitatively analyzed B-cell potential at the single cell level to determine the effect of lymphoid tissue site and antigen load on the expression of variable and constant regions. Concerning variable region expression, although the total frequency of B-cell precursors for phosphorylcholine is similar between nonimmune spleen and gut-associated Peyer's patch tissues, the proportion of cells producing non-TEPC 15 idiotypes is greater from Peyer's patch than from spleen. Oral immunization with phosphorylcholine-containing Ascaris suum increased the frequency of non-TEPC 15 B cells. Thus variation in the proportion of cells bearing different variable regions may be related to the distinct antigenic environment of cells in Peyer's patches compared to that of cells in spleen. Regarding constant region expression, although B cells from both spleen and Peyer's patches generate clones producing IgM, IgGl, and IgA singly and in all combinations, cells from Peyer's patches generate more clones secreting only IgA than cells from spleen. B cells specific for phosphorylcholine and inulin, which are found on intestinal bacteria, produce more IgA-only clones than B cells specific for the dinitrophenyl determinant. This striking correlation between IgA expression and variable region specificity for antigen implies that environmental antigens have expanded certain B cells in Peyer's patches which then have the ability to generate progeny that express only IgA. Evidence supporting the secondary nature of precursors for IgA-only clones is obtained by their ability to produce this isotype after stimulation with histoincompatible T cells. The role of gut antigens may be to clonally expand IgA precursors and perhaps to stimulate the proliferation of less differentiated cells within the unique microenvironment of the Peyer's patches, allowing them to differentiate to IgA precursors.

Rabbit lymphoid cells differentiated with respect to alpha-, gamma-, and mu- heavy polypeptide chains and to allotypic markers Aa1 and Aa2.

Lymphoid cells present in spleen and lymph nodes of hyperimmune rabbits were found to be differentiated with respect to the class of immunoglobulin heavy chain which they contained. The relative proportions of cells containing the various heavy chains were as follows: alpha-chain (5 to 8%), micro-chain (14 to 21%), and gamma-chain (71 to 81%). The allotypic markers Aa1 and Aa2, found on heavy chains, were also found to be separately localized in cells of Aa(1)/Aa(2) heterozygous rabbits. The ratio of cells in spleen and lymph nodes containing the Aa1 marker to those containing the Aa2 marker varied with individual rabbits; the range was 53 to 88% Aa1 versus 12 to 47% Aa2.

Special features of the priming process for a secretory IgA response. B cell priming with cholera toxin.

Administration of cholera toxin/toxoid by either intraduodenal or parenteral routes increases the frequency of antigen-sensitive B cells in Peyer's patches (PP) and in distant lymphoid tissues greater than 50-fold. The special feature of mucosal priming with toxin is its unique effectiveness at generating secondary B cells, whose progeny express IgA exclusively, and such cells appear in highest frequency in PP and in appreciable numbers in spleen. Thus, this deliberate intraduodenal immunization seems to mimic the natural priming process induced by enteric bacterial colonization, which we have postulated to account for the high frequencies of IgA-committed cells specific for bacterial determinants in the PP of conventionally reared mice. furthermore, as a result of intraduodenal immunization, antigen-specific memory B cells are disseminated to sites distant form that of antigen application, including the lymphoid follicles associated with the respiratory mucosa. Direct antigenic stimulation of cells in the PP therefore results in effective cross-priming among mucosal and systemic sites through division, differentiation, and disemination of antigen-sensitive secondary B cells.

Peyer's patches: an enriched source of precursors for IgA-producing immunocytes in the rabbit.

The proliferative and differentiative potential of Peyer's patch, peripheral blood, and popliteal lymph node cells was assessed by allogeneic cell transfer followed by quantitation of donor immunocytes by immunofluorescence. It was found that Peyer's patches are a highly enriched source of cells which have the potential to proliferate and differentiate into IgA-producing immunocytes and that the Peyer's patch cells are far more efficient in seeding the gut of irradiated recipient rabbits with donor cells that give rise to immunoglobulin-producing cells than cells from peripheral blood or popliteal lymph nodes.

Restriction of gene expression in B lymphocytes and their progeny. III. Endogenous IgA and IgM on the membranes of different plasma cell precursors.

Fluorescent antibody staining with antibodies to the f and g locus allotype markers present on rabbit alpha-chains revealed that the alpha-chain is the heavy chain on the Peyer's patch lymphocytes which previously had been shown to be the precursors of IgA-producing plasma cells. In addition, lymphocytes which had been stripped of membrane Ig with pronase and then cultured overnight to allow the sole expression of endogenous membrane Ig were found to have either the micro-chain or the alpha-chain on their membranes, but not both. These results suggest that most lymphocytes are restricted to the synthesis of one class of heavy chains at a time and that the commitment to synthesizing that particular heavy chain is maintained during the differentiation of lymphocytes into plasma cells. The proportion of lymphocytes with membrane alpha-chains is higher in the Peyer's patch and appendix, two gut-associated lymphoid tissues (GALT), than in other lymphoid tissues. Since the GALT are enriched sources of precursors for IgA-producing plasma cells compared to nongut-associated tissues, the presence of cells bearing membrane alpha-chains correlates well with the relative abilities of these tissues to generate IgA plasma cells.

Gut mucosal immunization with reovirus serotype 1/L stimulates virus-specific cytotoxic T cell precursors as well as IgA memory cells in Peyer's patches.

In this report we have shown that reovirus 1/L is an effective mucosal immunogen capable of generating a cytotoxic T cell (CTL) and associated helper T cell response to the nominal antigens associated with reovirus 1/L. The effectors that mediate reovirus-specific cytotoxicity are Thy-1+, Lyt-2+, and major histocompatibility complex (MHC)-restricted in their recognition of reovirus antigens, and can therefore be classified as CTLs. Frequency analysis of precursor CTLs occurring in Peyer's patches (PP) and peripheral lymph nodes (PLN) 6 d and 6 mo after intraduodenal stimulation have demonstrated that a persistent gradient of precursors is established, with higher frequencies present in PP. The generation of a CTL response in PP may be important in preferentially repopulating mucosal tissues with effector CTLs that could result in the local containment of infections in the gut. We also found that reovirus 1/L generates a virus-specific B cell response that is dominated by IgA memory cells after intraduodenal immunization. We hypothesize that the efficacy of reovirus 1/L at stimulating T and B cells in the gut mucosa is related to its ability to selectively enter PP via microfold (M) cells after enteric application. In this study we have also demonstrated that PP cells, upon in vitro culture and unrelated to prior reovirus priming, can generate natural killer-like (NK) cytotoxic activity. This may be an in vitro correlate of the in vivo generation of effectors that may populate mucosal tissues (i.e., the intestinal epithelium) with NK-like effector cells.

Relationship between expression of IgA by Peyer's patch cells and functional IgA memory cells.

IgA memory B cells have been operationally defined as precursors that give rise to clones exclusively secreting IgA antibodies upon antigen stimulation in a T-cell dependent splenic fragment culture. B lymphocytes that are sIgA+ account for a small fraction of Peyer's patch lymphocytes, but these can be clearly divided into two subsets. One subset contains the majority of sIgA+ B cells and most of these are in S, G2, or M phase of the cell cycle. These cells are germinal center B cells, as defined by being S kappa low and peanut agglutinin (PNA)high, and contain most of the mRNA alpha. Though these germinal center cells may contain the majority of sIgA+ B cells and may contain precursors for memory cells, preplasma cells, or both, they do not appear to be immediately responsive to stimulation by antigen. Rather, the S kappa high, PNAlow subset of sIgA+ B cells, most of which are in G0 or G1 and have only low levels of mRNA alpha appear to contain most of the clonal precursors that are committed to IgA, i.e., the functional memory cells that give rise to clones exclusively secreting IgA upon stimulation with thymus-dependent antigen in the presence of T cells. There is also a population of Peyer's patch B cells that neither bears detectable sIgA nor has mRNA alpha detectable by cytoplasmic dot blotting but contains a small proportion of the functional IgA memory cells.

Transport of immunoglobulins from serum into colostrum.

Oligomeric, J-chain-containing immunoglobulins were observed to be transferred selectively from serum into colostrum. These studies suggest that, in the case of the mammary gland secretion, a significant role for extraglandular synthesis of IgA merits consideration. Thus, for example, colostrum may contain antibodies synthesized locally as well as antibodies synthesized in the much larger lymphoid tissues such as the gut lamina propria.

Restriction of gene expression in B lymphocytes and their progeny. I. Commitment to immunoglobulin allotype.

Lymphocytes from b(5)/b(9) rabbits were stained in suspension with fluorescent antiallotype antibody reagents to selectively label with fluorescent molecules those cells bearing membrane immunoglobulin (Ig) of the b5 or b9 allotype. After staining, the cells were separated by the fluorescence-activated cell sorter into populations markedly enriched in cells bearing b5 or b9 membrane Ig or totally depleted of cells with detectable membrane Ig. The potential of these separated cells to give rise to Ig-synthesizing plasma cells either in vivo after transfer into irradiated recipients or in vitro during culture in the presence of phytohemagglutinin or pokeweed mitogen was assessed by immunofluorescence. The relative proportion of b5 and b9 cytoplasmic Ig-stained cells (CSC) arising from the separated cells was determined to test directly whether B lymphocytes and their progeny are committed to the synthesis of Ig of one allotype. It was found that b5- and b9-bearing cells gave rise almost exclusively to b5- and b9-producing plasma cells, respectively, in both the in vivo and in vitro assay systems. Most of these CSC were probably not derived from previously existing CSC but arose as the result of the differentiation of lymphocytes with membrane Ig. When cell populations totally depleted of Ig-bearing lymphocytes were cultured, very few CSC were found, indicating that the majority of immediate precursors of CSC have membrane Ig. These results suggest that individual B cell clones are phenotypically restricted to the expression of immunoglobulin genes on one chromosome; the significance of this clonal allelic exclusion is discussed.

Restriction of gene expression in B lymphocytes and their progeny. II. Commitment to immunoglobulin heavy chain isotype.

To determine whether or not B lymphocytes are committed to the synthesis of a single immunoglobulin heavy chain isotype during their differentiation into plasma cells, rabbit lymph node and Peyer's patch cells were separated into populations with and without membrane IgM, using a fluorescence-activated cell sorter (FACS). The potential of the micro-bearing (micro+) and non-micro-bearing (micro-) cells to give rise to plasma cells both in vivo after transfer into irradiated recipients and in vitro in the presence of pokeweed mitogen was assessed by immunofluorescence techniques, and the relative proportions of the cytoplasmic Ig-stained cells (CSC) synthesizing each class of heavy chains were determined. Most of the CSC arising in vitro from micro-bearing lymph node and Peyer's patch cells contained IgM; all IgM CSC appeared to be derived from micro+ cells. Peyer's patch lymphocytes, however, did not generate IgM CSC after cell transfer and thus may be functionally different from lymph node micro+ cells. It was found also that nearly all of the many IgA CSC generated by Peyer's patch lymphocytes either in culture or after transfer were derived from micro- cells. Further fractionation of these micro- cells with the FACS after they had been membrane stained with anti-b locus allotype reagents revealed that the precursors of IgA CSC belong to a minor population of cells which do have b locus light chain determinants on their membranes, although they do not have detectable micro-chains. These cells are not found in lymph nodes. Although the majority of Peyer's patch and lymph node cells were found to be precommitted to the synthesis of a single heavy chain isotype, a small proportion of cells may not be similarly restricted. Some of the CSC with membrane IgM were found to contain cytoplasmic IgA or IgG. In addition, micro+ populations did give rise to low numbers of IgA and IgG CSC. The implications of these results, obtained under experimental conditions, on the normal differentiation of B lymphocytes in situ are discussed.

Reovirus-induced liver disease in severe combined immunodeficient (SCID) mice. A model for the study of viral infection, pathogenesis, and clearance.

Adult severe combined immunodeficient (SCID) mice can be infected by the oral route with reovirus, and a systemic infection can be established. Infectious virus is recovered from all internal organs, and the mice die in 4-6 wk. Chronic, discrete inflammatory lesions appear in the liver of infected mice, and are associated with hepatocytes containing demonstrable levels of viral antigen. The adoptive transfer of Peyer's patch (PP) cells from congenic mice before infection protects the SCID mice against disease and death. Immune donor PP cells can be distinguished from nonimmune cells by their ability to contain and resolve infection by 1 wk after challenge.

Phosphorylation of lymphocyte myosin catalyzed in vitro and in intact cells.

Myosin has been isolated from guinea pig B-lymphocytic leukemia cells (L2C). The myosin has been enzymatically phosphorylated and dephosphorylated in vitro using both heterologous and lymphocyte-derived enzymes. Both the heavy chain and 20,000-dalton light chain of lymphocyte myosin are phosphorylated in vitro. Phosphorylation of myosin enhances actin-activated ATPase activity. Phosphorylation of myosin in murine lymphocytes was analyzed by use of a novel technique for rapid immunoprecipitation of myosin from cell extracts. Both the heavy chain and 20,000-dalton light chain of myosin are phosphorylated in intact cells. Addition of antibody reactive with cell-surface immunoglobulin to lymphocyte populations enriched for B cells stimulates locomotion of these cells and also increases the quantity of 32P isolated in association with the 20,000-dalton light chain of lymphocyte myosin, when 32Pi was present in the medium. In addition, an unidentified, phosphorylated polypeptides with a molecular mass of 22,000 daltons is co-isolated with myosin from cells by rapid immunoprecipitation. These results are consistent with the hypothesis that phosphorylation of myosin may contribute to regulation of movements performed by lymphocytes which are related to their participation in immunologic reactions.

Specificity of antibodies: primary structural basis of hapten binding.

The primiary structure of the 83 residues of the NH(2)-terminus of the V(II), region was determined for each of three different antibodies to hapten which were produced in inbred guinea pigs. Each antibody had a different and distinctive primary structure within each of the two "hypervariable" regions (Hv1 and Hv2) included in the analyzed part of the variable region of the heavy chain. The sequences of Hvl and Hv2 in the three antibodies were either unique or of restricted variability compared with those of "normnal" immunoglobulin G2. Further implication of Hv1 and Hv2 in contributing to ligand-binding specificity of antibodies came from the placement of residues modified by affinity labeling reagents in these hypervariable regions.

Selective simulation of allelic expression: effectf antibodies to allotypic markers on lymphoid cells.

Peripheral blood leukocytes from rabbits which were heterozygous (b(5)/b(9)) for markers on their immunoglobulin light chains were maintained in vitro for up to 24 hours in the presence or absence of antibody to b9. After culture they were transferred into lethally irradiated b(4)/b(4)hosts. Recipients of cells exposed to antibodies to allotype markers showed a striking increase in concentration of circulating b9 molecules and number of b9 plasma cells in their spleens compared pared to control animals receiving untreated cells from the same donor. There was no appreciable difyerence between the two groups of recipients with respect to their content of b5 molecules and immunocytes.

Quality of antibodies secreted by clones in microcultures from B cells enriched on haptenated gelatin: isotypes and avidities.

Populations of murine B cells enriched for fluorescein (FLU)- or phosphocholine (PC)-binding cells stimulated with LPS, or FLU- or PC-LPS at low density in 10 microliter cultures form clones of cells that secrete antibodies. Antibody isotypes were determined by radioimmunoassay and their avidities were determined relative to standard, monoclonal antibodies by hapten inhibition using a radioimmunoassay. These analyses further characterize the development of B cell clones in microcultures and reveal that differing culturing conditions stimulate qualitatively different B cell populations to divide and differentiate. Without filler cells, isotype switching is rare. Co-culturing B cells with 10(5) (CBA/N x BALB/c) F1 male thymocyte filler cells leads to IgG and/or IgA antibody secretion by 15-20% of cultures; antibodies from clones that switch isotypes are exclusively of high avidity. IgM is almost always present as one clonal product; pre-switched cells rarely score in microcultures. Without filler cells, a high percentage of antibodies from FLU-LPS stimulated, FLU-binding cells are of high avidity (60%). However, clonotypes of lower avidity dominate with mitogenic culture conditions, 100 micrograms/ml LPS or with thymocytes. PC-binding cells are less sensitive to these mitogenic effects. Antibodies produced by PC-specific clones have a more restricted pattern of avidities and resemble in quality anti-PC antibodies produced in vivo.

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.

Interrelationship of primed B cells with the potential for IgE and/or IgA expression.

The relationship between the pathways of B cell differentiation leading to IgE or IgA expression was analyzed by assessing the isotype potential of primed B cells as revealed over many generations of clonal outgrowth in splenic fragment cultures. Cells from (CBA/N X BALB/C) F1 male and female mice primed with phosphocholine (PC)-hemocyanin (Hy) and given a secondary stimulus with PC-Hy or PC-determinants via an Ascaris infection gave rise to a large proportion (25-48%) of clones which expressed anti-PC IgE along with any one or mixture of other isotypes, especially IgG and/or IgA. Accompanying the appearance of these cells in the Peyer's patches following Ascaris infection was the steady rise in IgA committed cells over a 12 week period. The potential to express IgE seems to be a normal feature of the development of secondary or memory cells. The coexpression of IgE randomly with all other isotypes supports a linear rather than a branched pathway of B cell differentiation. Ascaris or PC-determinants given to F1 mice were not unique in their ability to prime cells with the potential for IgE expression. Stimulation of BALB/c mice with two low doses of N-acetyl-glucosamine-conjugated hemocyanin (GlcNAc-Hy) primed cells in vivo generated a high proportion (63%) of clones in vitro that expressed IgE and most of these exclusively coexpressed IgA (16/26) suggesting a progressive restriction in isotype potential. Cells which gave rise to IgE producing clones specific for the priming hapten did not support the expression of IgE by clones of other specificities costimulated in vitro (anti-inulin, anti-beta-galactosyl). Thus the potential to express IgE seems to be both an inherent property of the B cells and under hapten-specific or hapten-linked regulation.

CH isotype switching in B cell differentiation.

We have functionally defined a number of B cell subsets that likely represent B cells at different stages of development, based on the pattern of CH isotypes expressed by their clones in splenic fragment or microcultures and on those factors necessary in culture to support the growth of a clone displaying a particular isotype or set of isotypes. Our observations are consistent with isotype switching being a stochastic process which results in the occurrence of progressive isotype restriction in members of a diversifying clone. The surface marker best predictive of the pattern of isotypes a clone may secrete is the sIg isotype of its B cell precursor. Those B cells that have switched to the expression of non-IgM isotypes in vivo can be stimulated in vitro in splenic fragments to give an antibody-secreting clonal culture but so far cannot be stimulated in a microculture of dispersed cells that supports clones secreting IgM alone or with other isotypes.

Influences of microbiota on intestinal immune system development.

The normal colonization of the mammalian intestine with commensal microbes is hypothesized to drive the development of the humoral and cellular mucosal immune systems during neonatal life and to maintain the physiologically normal steady state of inflammation in the gut throughout life. Neonatal conventionally reared mice and germ-free, deliberately colonized adult mice (gnotobiotic mice) were used to examine the efficacy of certain intestinal microbes.