Biomedical subjects

D Primi

Publications and source records attributed to D Primi.

At least 73 records · Page 4Linked to original sources

PubMed1987

Highly preferential VH-VL pairing in normal B cells results in antigen-independent selection of the available repertoire.

The aim of this work was to analyze the probabilities of combined expression of the various polymorphic forms of two well-defined VH and VK segments. The availability of two monoclonal antibodies specific, respectively, for the VHT15 and VK21 D-E gene products allowed us to study, both in the selected and in the preimmune repertoire, the expression of VHT15-VK21 D-E pairs in several mouse strains. Our data establish, first, that even before antigen encounters B cells, clones utilize a highly biased repertoire of VH-VL combinations and, secondly, that the level of productive VH-VL pairing depends primarily on the "allelic" form of the relevant VH and VK genes. Furthermore, clonal analysis revealed that this asymmetry in Ig gene expression affects a large proportion of newly arising B cells. Together, these studies demand reconsideration of the current estimates of the available antibody repertoire size, and provide new insights for our understanding of the phenomenon of clonal dominance.

Alleles↗

PubMed1986

Paucity of phosphorylcholine-specific clones in B cells expressing the VHT15 gene product.

The aim of this work was to study the cellular basis of the phenomenon of clonal dominance. To this end we analyzed two collections of BALB/c and C.B20 hybridomas that we selected on the basis of the expression of the VHT15 gene product independently from their antigen specificity. Our study demonstrates that none of the 28 BALB/c and only 2 of the 29 C.B20 hybridomas obtained have variable regions that bind PC. We conclude therefore that the domination of the immune response to PC by particular variable regions cannot be due to the establishment of clonal dominance prior to immunization.

Animals↗

PubMed1986

B cell triggering properties of a nontoxic derivative of amphotericin B.

The immunomodulating properties of amphotericin B (AMB), an antifungal polyene antibiotic, have been reported in multiple studies. However, many findings on the subject are conflicting, and the precise mechanism of AMB action on the immune system is yet unknown. Because toxicity and limited solubility of AMB are likely to be responsible for these discrepancies, we synthesized a nontoxic derivative of AMB (AMBSH), and we investigated its immune modulating effects on murine B cells. Our results show that AMBSH induces a strong proliferative response under conditions where AMB is weakly efficient or toxic, and that AMBSH supports maturation to Ig secretion. When suboptimal doses of LPS (or BCGF) are present together with AMBSH, a synergistic effect on B cell proliferation occurs. Frequency analyses reveal that, although only a limited number of B cells respond to AMBSH alone, a large population of B cells will respond to subthreshold doses of LPS in the presence of this polyene. Finally, we show that incubation of spleen cells with AMBSH results in an increase in Ia expression. These results are discussed in terms of the membrane disorganizing properties of polyene antibiotics.

Amphotericin B↗

PubMed1986

Lack of expression of the VHS107 gene family in the lipopolysaccharide-sensitive B cell subset of X-linked immunodeficiency-defective mice.

The expression of the VH T15 gene product was analyzed in the sera and in the supernatants of polyclonally activated B cells of X-linked immunodeficient (xid) mice. We found that defective males, contrary to normal females, do not express the VH T15 gene product even on antibodies devoid of anti-PC specificity. RNA analysis of polyclonally activated cells with VH specific probes revealed that xid-defective B cells do not express the entire S107 or part of the J606 VH family. Members of the J558 family, on the other hand, are equally well expressed among defective males and normal female B cells. These results strongly suggest that VH families are asymmetrically represented among B cell subsets.

Animals↗

PubMed1986

Differential expression of sets of highly homologous variable region gene products in selected and preimmune repertoires of inbred mouse strains.

Using mAb that selectively recognize the various allelic forms of the VHT15 and Vk21D-E genes' products, we analyzed the influence of VH and Vk polymorphism on the probability of expression of these gene segments. Our data show that the frequency to which the VHT15 gene product becomes available in the preimmune repertoire is strongly influenced by the polymorphism of the relevant structural gene, suggesting therefore that VH genes cannot be randomly used in the various strains. Contrary to this, the frequency of Vk21D-E+ clones is similar in all mouse strains tested, and in all cases is higher than the frequency of VHT15 clones. This observation strongly suggests that Vk genes can be randomly expressed, and/or that their number is lower than that of their VH counterpart. Finally, analysis of the specificity associated to the expression of the VHT15 segment revealed that VH polymorphism strongly influences not only the probability of expression of each V gene, but also the specificity of the antibodies on which these VH genes are used.

Animals↗

PubMed1986

The level of lambda 1 light chain expression in the mouse reflects the probability of rearrangement of the relevant gene.

The predominance of kappa over lambda light chain expression in mice can either reflect the probability of rearrangement of the relevant locus or be the result of antigen-driven clonal expansion. To discriminate between these two possibilities we determined, by limiting dilution analysis, the frequencies of kappa- and lambda-producing cells in B lymphocytes generated in vitro from bone marrow pre-B cells. The frequencies obtained in these cultures are not significantly different from those obtained with mature spleen cells. In addition, Southern blot analysis of bone marrow-derived and splenic cell DNA revealed that in both populations the extent of B lymphocytes having undergone lambda 1 gene rearrangement does not exceed 4%. These results, therefore, establish that in the mouse the low level of lambda light chain expression directly reflects the probability of rearrangement of the relevant locus.

Animals↗

PubMed1986

Structural polymorphism of V kappa 21 E and V kappa 21 D gene products in laboratory mice.

The aim of the present work was to study structural polymorphism of V kappa genes products. To this end we isolated immunoglobulins expressing the V kappa 21 D and V kappa 21 E gene products from the normal sera of several inbred strains of mice using a monoclonal antibody that selectively recognizes V kappa 21 D and V kappa 21 E subgroups. Analysis of the isoelectric focusing pattern of the light chain of these immunoglobulins revealed the existence of 3 clearly different phenotypes. The first one is shared by most inbred strains of mice, the second one is expressed by C58/J, AKR and PL/J and the third one defines V kappa 21 D-E light chains of SJL and SJA mice. Genetic analysis revealed that the locus controlling V kappa 21 D-E chain structure is closely linked to the Ly2-3 and to the Ig Kappa-Ef1 loci. Finally, using recombinant mouse strains, we could also order V kappa 21 D-E genes with respect to the known loci affecting V kappa polymorphism. Taken together our data argue against the possibility that the polymorphism observed results from strain-specific expression of V kappa genes common to all mice, but rather suggest that different allelic form of the same V kappa gene subgroup exist in different strains.

Animals↗

PubMed1986

The commitment of secretory cells to the selective expression of immunoglobulin CH genes is determined by the available concentrations of the triggering ligand.

The property of lipopolysaccharide to induce B cells to both proliferate and differentiate to IgM, IgG3 and IgG2b expression can be ascribed either to a precommitted sequence of molecular events in the activated B cells or, alternatively, to separate activities which independently modulate the two events. To discriminate between these two possibilities we have investigated the relationship between the doses of the polyclonal stimulus and the commitment of the activated cells to proliferate and to produce various isotypes. Low doses of ligand supported proliferation as well as IgM but not IgG2b secretion. On the contrary, high doses of the same ligand were less efficient in supporting proliferation but strongly induced heavy chain class switch. The effect of lipopolysaccharide concentrations on CH genes expression decayed with the distance from mu to the respective C gamma gene. Although we could define different B cell subsets on the basis of their proliferative response to various doses of the ligand, all these B subpopulations were found to be multipotential in terms of their switching capacity. Taken together our data show that in lipopolysaccharide cultures B cell proliferation and heavy chain switch are two events completely dissociable on the basis of their inducing requirements.

Animals↗

PubMed1986

Analysis of immunoglobulins expressing the V kappa 21E and V kappa 21D gene products. Evidence for multigenic control of the expression of VH-VL combinations.

The aim of this work was to study the genetic control of VH-VL combined expression. To this end immunoglobulins expressing the V kappa 21E and V kappa 21D gene products were isolated from the normal sera of several inbred strains of mice using a monoclonal antibody that selectively reacts with V kappa 21E and V kappa 21D subgroups. Analysis of the isoelectric focusing (IEF) pattern of the IgG heavy chains of these immunoglobulins obtained from H-2-congeneic mice revealed as expected that Igh-linked loci strongly influence VH-VL combined expression. The IEF pattern of V kappa 21D and V kappa 21E-associated heavy chains isolated from recombinant inbred mouse strains, however, revealed different phenotypes from either parental strains in strains in which both H-2 and Igh loci segregated from the same parent. These data, therefore, strongly suggest that the expression of VH-VL combinations is controlled in part by genetic loci which segregate independently from those linked to H-2 and Igh.

Animals↗

PubMed1985

The regulatory locus r lambda 1 affects the level of lambda 1 light chain synthesis in lipopolysaccharide-activated lymphocytes but not the frequency of lambda 1-positive B cell precursors.

Several strains of mice, most notably the SJL strain, have a greatly reduced level of circulating lambda 1 immunoglobulins (r lambda 1 lo phenotype) compared with other mice. The locus responsible for this phenotype has been shown to be closely linked to the structural C lambda 1 gene. Functionally this locus has been said to reduce the number of lymphocytes expressing surface lambda 1 molecules. In order to gain a better understanding of this phenomenon we compared the functional properties of activated B cells secreting lambda 1 immunoglobulins in the splenocytes of both BALB/c and SJL mice. Our results indicate that regulatory T cells, as well as regulatory ontogenetic processes, are not responsible for the r lambda 1 lo phenotype. In addition, limiting dilution analyses revealed that the number of lipopolysaccharide-sensitive precursors of lambda 1-secreting B cells was similar in the splenocytes of the two strains of mice tested. The quantity of lambda 1 molecules produced by a B cell clone, however, was found to be lower in SJL than in BALB/c mice. As the level of lambda 1 mRNA is greatly reduced in lipopolysaccharide blasts of SJL mice, as compared to the mRNA detected in BALB/c blasts, we conclude that the impairment responsible for the r lambda 1lo phenotype is probably transcriptional. We tentatively propose that sequences 5' to the C lambda 1 region are defective in their capacity to enhance the lambda 1 transcripts in SJL mice.

Animals↗

PubMed1984

The B cell repertoire revealed by major histocompatibility complex-specific helper T cells. I. Frequencies of a genetically defined V region marker among mitogen- and T helper cell-reactive B lymphocytes in normal and immunized mice.

The aim of the present work was to analyze the frequencies of a genetically defined variable (V) region marker in the B cell subset sensitive to T cell help. To this end we used an alloreactive T cell line that has the property of inducing B cells of the appropriate haplotype to exponential growth and polyclonal antibody synthesis. The frequency obtained with this helper line was also directly compared to that obtained with lipopolysaccharide (LPS). We found that in normal BALB/c mice the frequency of M460-positive clonotypes was respectively, 1/100 and 1/1,000 among the T helper- and LPS-sensitive B cell subsets. In mice immunized with antiidiotype coupled to a thymus-dependent antigen, the differences in the numbers of idiotype-positive precursors were even more accentuated, i.e. 1/20 in the B cell subset triggered by T helper cells and 1/800 in those cells responsive to LPS. The frequencies of the M460 determinant in mice immunized with anti-idiotypes coupled to thymus-independent antigens were not significantly different, in either B cell subset, from those obtained with spleen cells of normal nonimmunized animals. Taken as a whole, our results imply that the V gene repertoire revealed by LPS includes precursor distribution, as this distribution occurs during the early stage of B cell development (potential repertoire), while the repertoire revealed by T helper cells includes the V region distribution of those clones that are selected in the periphery of the functional immune system.

Animals↗

PubMed1984

Selective and polyclonal induction of high levels of lambda light chain-bearing immunoglobulins in BALB/c and SJL mice.

Polyclonal B cell activation is V gene independent and consequently results in the unselected expression of both V lambda and V kappa genes. In an attempt to selectively and polyclonally trigger lambda light chain-bearing B cells, we immunized mice with low doses of lipopolysaccharide (LPS) coupled to antibodies specific for the constant region of lambda 1 chains. Analyses of the sera of treated mice revealed that LPS anti-lambda 1 selectively triggers lambda-but not kappa-bearing B cells to high rate Ig secretion. Surprisingly, immunized SJL mice (rlo lambda 1 phenotype) mounted a lambda + response comparable to that detected in mice with the r + lambda 1 phenotype. Because we could definitively establish that the majority of the induced antibodies belong the lambda 1 subtype, our results have strong implications both for the study of the repertoire of lambda 1-bearing B cells and for our understanding of the functional mechanism(s) by which the r lambda 1 locus acts.

Animals↗

PubMed1984

Dextran sulphate selectively enhances interleukin-2-dependent cytotoxic T lymphocyte growth but not differentiation.

We have recently observed that dextran sulphate (DxS) interacts with molecules responsible for terminal B-cell maturation. Since this property of DxS is most likely due to its anionic characteristics, we have investigated whether this molecule could also modulate the response of cytolytic T cells (CTL) to interleukin-2-conditioned medium (IL-2 CM). Our data demonstrate that DxS synergizes with IL-2 in supporting the clonal expansion of CTL. Using limiting dilution analysis, we were able to establish that DxS acts directly on IL-2 CM and not on the responding lymphocytes and that the synergism observed in the result of increases in both clone size and the frequency of CTL precursors. Finally, since we could also demonstrate that DxS selectively affects the growth- but not the differentiation-promoting activity present in IL-2 CM, our results lend further support to the notion that CTL growth and differentiation are mediated by distinct soluble products. The possible mechanisms by which DxS alters the property of CTL growth-supporting activity are discussed, together with the practical implication of our findings.

Animals↗

PubMed1983

The selection and maintenance of the V region determinant repertoire is germ-line encoded and T cell-independent.

The number of lipopolysaccharide-sensitive precursor cells synthesizing immunoglobulin (Ig) which reacts with the monoclonal anti-M460 antibody F6(51) has been determined in the spleen and in the bone marrow of different strains of mice. These precursor frequencies fall into two quantitatively different groups. The first group includes mice with the same Igh haplotype as BALB/c animals (Igha). In this group, spleen cells contained between 1:10(4) to 1:5 X 10(4) B cell precursors secreting Ig which bound F6(51). The second level of precursor was obtained with animals with allotypic haplotypes other than Igha. These values were too low to allow accurate frequency determinations. The frequency of these cells in mice of the latter group, however, increased dramatically when these animals were hyperimmunized with the monoclonal anti-M460 antibody. Similar results were obtained when the frequencies were determined using the Ig- fraction of bone marrow cells. Surprisingly, the numbers of lipopolysaccharide-sensitive B cell precursors secreting F6(51)-binding Ig in spleen cells of nude mice was found to be similar to the one in splenocytes of normal mice, and even in this case, the frequencies reflected the genetic background of the animals tested. Taken together these data support the notion that the establishment and the maintenance of the M460 idiotypic repertoire is germ-line encoded and independent of regulatory T cells.

Animals↗

PubMed1983

The simultaneous antagonistic effects of a T cell hybridoma product on the growth and the maturation of activated lymphocytes.

Lymphocyte maturation and growth are two antagonistic events; therefore, successful antibody synthesis accompanied by clonal expansion, as seen in most humoral immune responses, must be the result of a delicate quantitative balance between maturation and growth factors. On the basis of this hypothesis, it should be feasible to search for a T cell product that alters this equilibrium and favors either B cell growth or maturation. In an attempt to isolate T cell hybridomas producing these activities, we fused BW5147 thymoma cells with Con A-activated spleen cells. One of the hybridomas obtained constitutively produces a product (or products) that selectively inhibits mitogen-induced replication but not maturation of normal but not transformed B lymphocytes. More importantly, the same product(s) supports Ig synthesis but not growth of LPS blasts, which suggests that we are dealing with a B cell maturation factor. The effect of this supernatant can be completely abrogated by the B cell mitogen DxS. In addition, the proliferative response of B cells to this ligand is unaffected by the hybridoma product. The implications of our results for understanding the mechanism of B cell triggering are discussed.

Animals↗

PubMed1982

The relationship between variable region determinants and antigen specificity on mitogen reactive B cell subsets.

On the basis of previous frequency determinations we could set up large numbers of cultures, each containing less than one competent precursor B cell specific for beta-galactosidase or for each of three idiotopes previously found on a monoclonal anti-beta-galactosidase antibody. Cultures were polyclonally activated by either lipopolysaccharide or Nocardia-delipidated cell mitogen. Each culture supernatant was individually tested for hemagglutination activity against sheep erythrocytes coupled with beta-galactosidase or with each of the three purified monoclonal anti-idiotypic antibodies. The results showed that only a minority of those clones positive for only one or two idiotopes recognized antigen. However, all those clones simultaneously positive for the three V region determinants recognized beta-galactosidase. The implications of these results for our understanding of the relationship between the antigen-binding site and idiotope expression are discussed.

Animals↗

PubMed1982

Mitogen-reactive B cell subpopulations selectively express different sets of V regions.

The experiments presented here were designed to investigate whether the idiotypic repertoire is equally distributed among B cells subpopulations as defined by mitogen reactivity. To this end we used lipopolysaccharides (LPS) and Nocardia delipidated cell mitogens (NDCM), which are two mitogens that have been described to act on different B cell subsets. The repertoire can be defined in quantitative terms as the frequency of B cells that are precursors for clones secreting immunoglobulin with a given specificity or with a determinate idiotype. We determined, therefore, the absolute frequency of LPS- and NDCM-sensitive B lymphocytes secreting immunoglobulin molecules that bear three idiotopes originally found on a monoclonal anti-beta galactosidase antibody. Because the frequencies of B cells carrying one of these idiotypes are dramatically different in the LPS- and NDCM-sensitive B cells subsets, we conclude that the idiotypic repertoire is not randomly distributed among mitogen-reactive B cell subpopulations.

Animals↗

PubMed1982

Idiotype regulation: evidence for the involvement of Igh-C-restricted T cells in the M-460 idiotype suppressive pathway.

Allotypic restriction of idiotype suppression has been shown in several idiotypic systems. It has also been demonstrated that naive BALB/c mice contain naturally occurring suppressor T cells capable of inhibiting the M-460 idiotype expressed on BALB/c B cells, and that T cells from allotype-congenic mice have no effect on this expression. Since we have previously shown that upon polyclonal activation B cells of all mice tested are capable of secreting M-460-positive anti-2,4,6-trinitrophenyl (TNP) antibodies, we postulated that idiotype suppressor cells may recognize idiotypes in conjugation with products of the Igh loci. In the present study we tested this hypothesis. The data clearly showed that regardless of their allotypic haplotype all strains of mice do possess suppressor cells capable of recognizing the M-460 idiotype. However, mixing experiments indicated that the functional potential of these suppressor cells can only be expressed when T and B lymphocytes come from allotype-matched animals. Efficient suppression in those cultures containing allotype-matched but incompatible B and T cells at the I-J region of the H-2 locus could be detected. Finally, using allotypic recombinant strains of mice, we found evidence that the restrictive elements in idiotypic suppression are the product of genes mapping in the Igh-C locus, or closely linked to it.

Animals↗