Frequency analysis of immunoglobulin V-gene expression and functional reactivities in bone marrow B cells

Frequency of immunocompetent B cells in bone marrow has been determined in vitro under culture conditions that allow the development in vitro under culture conditions that allow the development of every growth-inducible B cell into a clone of IgM-secreting PFC. Three limiting dilution culture systems were employed: a specific helper assay with SRBC as antigen and using activated T helper cells, a nonspecific helper assay using Con A-induced factors as a source of help, and polyclonal activation with LPS. From unseparated, normal C57BL/6J bone marrow 1 in 2200 to 1 in 2820 B cells were induced to form a clone of PFC to SRBC in each of the 3 systems. This corresponds to a frequency of 1 SRBC-specific clone in every 900 IgM-secreting LPS-reactive clones. The frequencies of specific plaque-forming B cell clones in terms of LPS-reactive B cells was 1 in 36 for NNP1-SRBC, 1 in 58 for TNP30-SRBC, 1 in 75 for NIP1-SRBC, and 1 in 230 for TNP3-SRBC. These frequencies of v-gene expression in bone marrow B cells are of the same magnitude as the corresponding frequencies for splenic B cells. Bone marrow B cells are also fully susceptible to stimulation by antigen in combination with either specific or nonspecific T cell help, as well as polyclonal activation by LPS, since every 3rd Ig-positive cells in marrow could be induced to form a clone of IgM-secreting cells. There is thus no difference in immunocompetence between surface Ig-bearing B cells from bone marrow and spleen.     

Cloning of murine transformed cell lines in suspension culture with efficiencies near 100%

Addition of 3 × 10⁶ thymus cells from either syngeneic, allogeneic or xenogeneic animals increases the cloning efficiencies of murine thymomas (EL-4, WC-2), B-lymphomas (McPC 1748, 38C-13), Abelson-virus transformed cell lines (F and K), mastocytomas (P815), myelomas (AbPC22, X63-AG8, 5563, MOPC 104 E, RFC 5, W 3469) and hybrids of myelomas and normal B-lymphocytes (Sp-1), all adapted to tissue culture, to near 100%. Thymus cells also increase the efficiencies of growth initiation in primary in vitro cultures of myeloma tumor cells (S117) transplanted in vivo, and of cells fused between the azaguanine-resistant X63-AG8 myeloma cell line and normal, LPS-stimulated B-lymphocyte blasts.     

Frequency analysis of functional immunoglobulin C and V gene expression in murine B cells at various ages

The frequencies of lipopolysaccharide- (LPS) reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of mice at different ages. A limiting dilution culture system was employed that allows the growth and development of every LPS-reactive B cell into a clone an IgM-secreting cells that are capable of switching to other Ig heavy chain isotypes (C gene expression). The secretion of IgM and IgG1 was assessed in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V gene expression) were detected with the use of plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The frequencies of LPS-reactive B cells in the spleen and BM of C3H/Tif, C57BL/Ka, BALB/c, and CBA/Rij mice appeared to be similar in 6- to 12- and 100-wk-old animals, as was the switch frequency to IgG1 secretion in three strains tested. Moreover, no age-related changes were observed in the frequencies of antigen-specific B cells within the pool of LPS-reactive B cells in the spleen and BM of C57BL/Ka mice. The frequencies ranged from 1 in 10 to 1 in 20 for NIP4- and NNP2-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 4000 for SRBC, HRBC, and GRBC. The specificity repertoire of the "spontaneously" occurring ("background") IgM-secreting cells in the spleen and BM, on the other hand, did differ between young and old C57BL/Ka mice. During aging the frequencies of the tested specificities decreased in the spleen but increased in the BM. Our data indicate that in unintentionally immunized mice the clonal selection of B lineage cells by antigen takes place at the level of the mature, antigen-reactive B cell.     

The effect of x-rays on the precursors of antibody forming cells (B cells) as measured with the in vitro limiting dilution assay

The effect of X-irradiation upon murine antibody-forming cell precursors (B cells) was established in cultures of spleen cells stimulated with the B cell mitogen lipopolysaccharide (LPS). At day 5 and 7 the numbers of IgM- and IgG2-secreting cells were determined in cultures of irradiated and nonirradiated spleen cells. From these numbers a Do of 0.6-1.2 Gy for the IgM, and of 0.9-2.1 Gy for the IgG2 response was calculated. Similar Do values were obtained under limiting dilution culture conditions. In the limiting dilution assay the effect of irradiation upon the size of the IgM-producing clones could also be determined. It was found that irradiation reduced the number of LPS-reactive B cells without affecting the size of the clones produced by the surviving cells.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.     

Increase of precursor frequency and clonal size of murine IgE-secreting cells by IL-4

IL-4 is able to preferentially enhance murine IgE levels in the supernatant of LPS-stimulated T cell-depleted splenic B cell cultures. Clonal and quantitative analysis of this response revealed that this is due partly to a 14-fold increased IgE precursor frequency and partly to a three-fold increased clone size of IgE-secreting cells. IL-4 increased the precursor frequency and the clone size of IgM-secreting cells not more than twofold. Both the IgM and IgE response in LPS-stimulated B cells were completely inhibited by the addition of anti-IgM mAb (M41) to the cultures, indicating that the IgE-secreting clones developed as subclones from precursors that express IgM. These cells lacked expression of membrane-bound IgE up to day 5 of the culture. Application of feeder cells in these cultures resulted in an increased precursor frequency of IgE-secreting clones among LPS-reactive B cells that is due, partially, to IL-4 produced by the feeder cells.     

Differential regulation of activation, clonal expansion, and antibody secretion in human B cells

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.     

Replacement perfusion of cultured eucaryotic cells: A method for the accurate measurement of the rates of growth,protein synthesis,and protein turnover

The fractional rates of protein synthesis (k_s) and degradation (k_p) were studied in the myeloma cell line SP2/0-AG14 grown at different rates (k_g). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific-radioactivity of labeled L-leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium-pulse labeling kinetics and pulse-chase kinetics. The rate of protein degradation was determined from the established relationship k_g = k_s – k_p. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (k_g = 0.017/hr) turnover represented a major fraction of total synthesis (k_p = 0.032/hr = 0.65ks). At rapid growth rates (k_g = 0.058/hr) the value of k_p was less than 0.005/hr. No major difference was observed between the k_s determined for individual cellular proteins (separated by SDS-polyacrylamide (7.5%) gel electro-phoresis) from rapid- and slow-growing cultures. Thus, with an invariable k_s, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging then changes in the growth rate of SP2/0-AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and WUS totally inhibited by addition of lcupcptin (10 μM) and pepstatin (2 μM), the specific inhibitors of lysosomal cathepsins B (L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0-AG14 appearsto be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH ₄Cl (10 mM) is added to cells growing with a k_g of 0.018/hr ± 0.001 (k_s = 0.050/hr ± 0.002) the growth rate increased to 0.051/hr ± 0.002 without change in the rate of protein synthesis (k_s = 0.049/hr ± 0.003). It is suggested from our data that the cellular growth rate of SP2/0-AG14 is regulated by the lysosomal apparatus; whether this regulation is itself regulated by either a specific compartmentalization of the lysosomal proteinases and/or their substrates or by endogenous protease inhibitors, should prove to be an exciting area for future investigation.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Enhanced hybridoma production by electrofusion in strongly hypo-osmolar solutions

Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids. The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature. Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l. Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8. Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory. The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber). This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion. It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures. At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied. Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion. Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession. Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established. This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process. The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins. Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.     

Functional characteristics of Peyer's patch lymphoid cells. IV. Effect of antigen feeding on the frequency of antigen-specific B cells.

The frequency of B cells in Peyer's patches from normal BDF(1) and sheep red blood cell (SRBC)-fed BDF(1) mice that could respond to antigenic determinants on SRBC and trinitrophenyl (TNP) was determined using an in vitro system of limiting dilution analysis. In normal mice, one B cell in 1.9 x 10(4) Peyer's patch cells could be induced to an anti-SRBC response and one B cell in 3.6 x 10(4) Peyer's patch cells could be induced to an anti-TNP response. The frequency of B cells capable of responding to SRBC in normal mice was similar in Peyer's patches and spleen. However, after feeding mice SRBC for 3 weeks, there was a 6-fold reduction in the frequency of B cells in Peyer's patches capable of responding to SRBC but no change in the frequency of B cells capable of responding to TNP. The average clone size of Peyer's patch B cells responding to SRBC was similar in normal and SRBC-fed mice. Although antigen-feeding does not stimulate Peyer's patch B cells in situ to humoral antibody synthesis, antigen-feeding can markedly alter the reactivity of the antigen-sensitive cell population in Peyer's patches. We previously demonstrated that T cells in Peyer's patches could be specifically carrier primed for helper function by SRBC feeding. We have now demonstrated that antigen-feeding reduced significantly the frequency of B cells in Peyer's patches capable of responding to the fed antigen. Peyer's patches appear to serve an important function as a sampling site for intestinal antigens.     

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.     

Induction of suppression by a murine nonspecific suppressor-inducer cell line (M1-A5)

A murine nonspecific suppressor-inducer cell line (M1-A5) was established by the limiting dilution method from the spleen cells of a mouse bearing an advanced methylcholanthrene-induced fibrosarcoma. Indirect immunofluorescence studies demonstrated that the M1-A5 cells were Thy-1-, sIg-, Ly-5+, MAC-, and 45% asialo GM1+. The M1-A5 cells were able to activate suppressor cells from unprimed, syngeneic normal spleen cells. These activated cells inhibited antibody production by cocultured syngeneic lymphoid cells. Induction of suppression by the M1-A5 cells was via the release of a suppressor-inducing factor, which was found to be protein in nature. Kinetic studies showed that when M1-A5 cells were separated from NSC by a dialysis tubing in Marbrook vessels, the M1-A5 cells required a minimum of 8 hr incubation period before suppressor cell activity could be demonstrated in precursor cells. On the other hand, induction of suppression by the suppressor-inducing factor required a minimum of 3 hr exposure of the precursor cells to the factor.     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Cloned lines of human cytotoxic T lymphocytes with specificity for influenza virus

The generation of human cytotoxic T cell clones with specificity for influenza virus and some of their characteristics are described. The clones were generated by limiting dilution of peripheral blood lymphocytes after two in vitro stimulations with autologous influenza A/USSR virus-infected cells and were grown in T cell growth factor. The majority of the virus-specific clones showed cross-reactivity for different influenza A virus subtypes but did not recognize influenza B virus-infected cells. The HLA specificity of two clones was further analyzed. One clone, LL33, was specific for HLA-Bw60, the other, clone WH5, for HLA-A1. Clone WH5 also seemed to recognize the serologically related HLA-A26 as restriction element for the recognition of the viral antigen. Whereas the virus-specific CTL clones had the OKT3+,4-,8+ phenotype, another clone, WH 49, exhibiting natural killer-like activity, was found to have the OKT3+,4+,8- phenotype.     

Mechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine

B lymphocytes require a source of T cell-like help to produce antibody to T cell-dependent antigens. T cell-derived lymphokines and C8-substituted guanine ribonucleosides (such as 8-mercaptoguanosine; 8MGuo) are effective sources of such T cell-like help. Addition of T cell-derived lymphokines to antigen-activated B cells together with 8MGuo results in synergistic B cell differentiation, amplifying the sum of the individual responses twofold to four-fold. Lymphokine activity is required at initiation of culture for optimal synergy with 8MGuo, whereas the nucleoside can be added up to 48 hr after the lymphokines with full synergy. 8MGuo provides a perceived T cell-like differentiation signal to B cells from immunodeficient xid mice, thereby distinguishing a subset of Lyb-5- nucleoside-responsive B cells from those activated by soluble anti-mu followed by B cell stimulatory factor-1, interleukin 1, and B cell differentiation factors, which are Lyb-5+. Moreover, at least a subset of the B cells recruited by the synergistic interaction of lymphokines and nucleoside is distinct from that responsive to 8MGuo + antigen, insofar as Sephadex G-10 nonadherent xid B cells fail to respond to either 8MGuo or lymphokines alone, but do respond to the combination. A distinct subpopulation can also be demonstrated among normal B cells by limiting dilution analysis in which the precursor frequency of antigen-reactive B cells in the presence of lymphokines or nucleoside alone increases substantially when both agents are present together. In concert with the kinetic data, these observations suggest that synergy derives at least in part from the ability of lymphokines to induce one or more elements the absence of which limits the capacity of a distinct B cell subpopulation to respond to 8MGuo.     

An analysis of B cell memory. I. Interaction of helper and suppressor effects in the in vitro expression of IgM memory.

The pfc response of Srbc primed IgM memory cells has been characterized by limiting dilution analysis in vitro, in which LPS was used to maximize the response of spleen cells to Srbc. The analysis suggested that, even under these conditions, expression of B cell memory was not directly assayed and cell collaboration effects were still basic to the system.Two types of cells, as defined by function, appeared necessary to elicit optimal clonal proliferation of IgM B memory cells: firstly, helper T cells were essential for B cell proliferation even with LPS present in culture. Under appropriate conditions, helper activity could be provided by normal thymus cells. Secondly, activated T cells were required for the maximal conversion of normal thymus cells to helper cells. A third activity, T cell-dependent suppression, was observed at high cell doses. The implications of these results and the need for a comprehensive analysis of in vitro conditions for each individual type of experiment is discussed.     

Mature T cells generated from single thymic clones are phenotypically and functionally heterogeneous

A limiting dilution system for cloning thymic CFU (CFUt) from murine bone marrow has been critically evaluated to test the clonal origin of the thymic colonies. Simultaneous limiting dilution transfer of three populations of bone marrow, each expressing a unique allelic cell surface determinant, resulted in independent segregation of donor-derived thymocyte populations within groups of recipient mice. Statistical analysis of the data allowed an estimate of 1 CFUt/3.3 x 10(4) i.v. transferred bone marrow cells. A pulse-chase experiment was utilized to establish whether CFUt seed directly to the thymus, or whether thymic seeding is secondary to extra-thymic engraftment. The results supported the conclusion that bone marrow CFUt utilize a specific interaction with thymic blood vessel endothelial cells to recognize and enter the thymus, and that this seeding occurs within 4 h of i.v. infusion. A kinetic analysis of emigration of the CFUt progeny into the peripheral blood revealed that, in most cases, an early wave of predominantly CD4+ CD8- lymphocytes emerges from the thymus approximately 4 wk after radiation and reconstitution. In a few cases, the first progeny of CFUt to emerge from the thymus were predominantly CD4- CD8+. Commitment of CFUt to TCR beta-chain rearrangements was assessed by quantitating expression of the V beta 8 family of TCR V region genes. Although some clones expressed a significantly higher or lower percentage of V beta 8+ cells, these differences were not stable with time. Thus, CFUt do not undergo absolute commitment to cell surface phenotype of TCR rearrangement, as reflected by the phenotypes of their progeny. Clones of mature peripheral progeny of CFUt could be expanded in culture in the presence of mitogen and growth factors; approximately 30 to 50% of proliferating clones could mediate cytotoxicity in a lectin-dependent assay, further indicating that CFUt are not absolutely committed to a particular T cell function.