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N R Klinman

Publications and source records attributed to N R Klinman.

At least 91 records · Page 5Linked to original sources
PubMed1978

The diversity of the influenza-specific primary B-cell repertoire in BALB/c mice.

The primary immune response of BALB/c mice to influenza (PR8) hemagglutinin (HA), a complex protein antigen, has been examined by the splenic focus assay, and the resulting monoclonal anti-HA antibodies have been characterized by their reactivity with heterologous viruses. The analysis of the primary B-cell response to HA revealed marked differences from responses previously defined for haptenic determinants. There were following differences: (a) the frequency of HA-specific B cells in both conventional and germ-free BALB/c mice was 1 in 1.0-1.5 X 10(5) splenic B cells, which is substantially lower than the frequency of B cells responsive to various simple haptenic determinants; (b) monoclonal anti-HA antibodies were predominantly of the IgA or IgM isotypes instead of IgG, which dominates antihapten responses; and (c) after immunization, the frequency of anti-HA-specific B cells increases by 10- to 50-fold, which is much greater increase than that observed after immunization with haptenic determinants. Fine specificity analysis of primary monoclonal HA-specific antibodies revealed extensive diversity and a considerable overlap with the specificities obtained from immune mice. Given the low overall frequency of HA-specific B cells, it could be calculated that the representation of most HA-specific clonotypes within the B-cell repertoire could not exceed 1 in 10(7) B cells. These findings indicate that the primary B-cell clonotype repertoire is extremely diverse and largely antigen independent in its generation.

Animals↗
PubMed1978

Differential expression of an equivalent clonotype among BALB/c and C57BL/6 mice.

The primary anti-phosphorylcholine (PC) response in BALB/c, C57BL/6, and congenic and recombinant inbred strains of these parental types has been examined in the splenic focus system. The frequencies of PC-specific precursors were shown to vary among these strains from 2 to 20 precursors per 10(6) splenic B cells. The distribution of these frequencies suggests that elements closely linked to or within the major histocompatibility complex may play a role in the determination of this parameter, although additional experiments are necessary to adequately assess this possibility. Moreover, all strains tested, regardless of immunoglobulin allotype, expressed monoclonal antibodies indistinguishable from the TEPC 15 myeloma protein (T15) clonotype. Further, the frequency of this clonotype in a given strain did not appear related to allotype, since both high and low T15 frequencies were found among strains of either the BALB/c (a(1)) or C57BL/6 (a(2)) allotype. The examination of normal serum for the T15 idiotype, however, revealed that only mice of the BALB/c allotype (a(1)) expressed the T15 idiotype in detectable quantities. After immunization with Diplococcus pneumoniae, sera from mice of the a(1) allotype consistently contained large quantities of the T15 idiotype, whereas sera from mice of the a(2) allotype exhibited various degrees of cross-reactivity with anti-T15 antibody. These results suggest that: (a) the allotype of an individual, although closely related to serum levels of an idiotype, is unrelated to the proportion of the precursor population which expresses that idiotype and; (b) the serum expression of a given idiotype may reflect regulatory processes, which act either during or before antigenic stimulation, rather than the actual clonotype representation in the repertoire. These findings indicate that distinctions must be made between the expression of idiotypic determinants within precursor B-cell populations and elements which regulate the subsequent appearance of those idiotypes in serum antibodies.

Animals↗
PubMed1977

Expression of phosphorylcholine-specific B cells during murine development.

The TEPC 15 (T15) clonotype, a putatively germline antibody specificity, does not appear in the neonatal B-cell repertoire until approximately 1 wk of age. This report extends this observation by the demonstration that (a) the T15 clonotype follows similar kinetics of appearance in germfree as well as conventionally-reared mice; (b) maternal influences and genetic background play a minor role in the development of the T15 clonotype since CBFI neonates raised by C57BL/6 or BALB/c mothers acquire the T15 clonotype at the same time in ontogeny as BALB/c neonates; (c) the lack of phosphorylcholine (PC)-specific B cells shortly after birth is reflected in a dearth of PC-binding cells in the neonate as well; and (d) no PC-specifc B cells are found in 19-day fetal liver or in bone marrow until 7 days of life, coincident with their appearance in the spleen. These findings, along with a previous report that PC-specific splenic B cells are tolerizable as late as day 10 after birth, confirm the invariant, late occurrence of the T15 clonotype and support a highly- ordered, rigorously predetermined mechanism for the acquisition of the B- cell repertoire. The results are discussed in light of other studies on the ontogeny of B-cell specificity, and in terms of the implications on the mechanism by which antibody diversity is generated.

Animals↗
PubMed1977

Antibody-specific immunoregulation.

In recent years, much evidence has accumulated which demonstrates that an animal's immune system has the capacity to recognize its own antibody idiotypes. These findings suggest that self-idiotypic recognition may potentially play a role in the regulation of B-cell responses. The experiments presented in this report were carried out to determine if an animal develops the ability to specifically regulate the synthesis of antibodies specific for an antigen, subsequent to primary immunization to the particular antigen and concomitant with an initial antibody response. Employing the splenic fragment culture system we have compared the response of primary donor B cells in irradiated recipients which have been previously immunized to hemocyanin (Hy) alone or dinitrophenyl (DNP)-Hy plus Hy. The results indicated that only 25-30 percent of DNP- specific B cells stimulated by DNP-Hy in Hy immunized recipients could bestimulated by DNP-Hy in recipients immunized with Hy as well as DNP-Hy. B-cell responses to other haptens, such as fluoresceinated-Hy, and secondary DNP-specific B-cell responses were unaffected in DNP-Hy immunized animals. The nontrivial and specific nature of the observed decrease in primary DNP-specific B-cell responses was verified by the finding that the response of CB20 donor cells, which differ from BALB/c mice only in the immunoglobulin heavy chain allotype-linked locus, was unaffected in BALB/c recipient mice which had been immunized with DNP-Hy. Thus, it appeared that during a primary humoral immune response to a T- dependent antigen, an antibody-specific regulatory mechanism is induced which specifically limits the stimulation of hapten-specific primary, but not secondary, B cells. The important implications that these findings have for the understanding of the control of primary B-cell responses and the generation of secondary B cells is discussed.

Animals↗
PubMed1977

In vitro tolerance induction of neonatal murine B cells as a probe for the study of B-cell diversification.

The susceptibility to in vitro tolerance induction has been implicated as a characteristic of B cells early in their development, since DNP-reactive B cells are tolerizable only during the first days after birth, and 25% of adult bone marrow cells are tolerizable. In the present study, a modification of the in vitro splenic focus technique was utilized to determine if PC-specific B cells, by virtue of their late expression (approximately 1 wk post-parturition), also display susceptibility to tolerance induction. The results demonstrate that at 7-10 days after birth, when over 90% of the DNP-specific splenic B cells are resistant to tolerance induction, the majority of PC-specific B cells are tolerizable. These results re-emphasize tolerance susceptibility as a characteristic of developing clones, confirm the late acquisition of PC-specific B cells, and support the contention that the acquisition of the specificity repertoire is a highly ordered, specifically predetermined process which is independent of antigen-driven events.

Animals↗
PubMed1977

The monoclonal anti-phosphorylcholine antibody response in several murine strains: genetic implications of a diverse repertoire.

The idiotypic identification of monoclonal antibodies has been used to define and enumerate clonotypes within the murine repertoire of B cells specific for phosphorylcholine (PC). The response in the BALB/c strain is dominated by a single antibody specificity which is identical to TEPC 15 protein; however, antibody without the TEPC 15 idiotype appears heterogeneous by idiotypic cross-reactivity and hapten inhibition of binding to antigen. Dissection of the PC-specific repertoire in the AKR, A/He, and C3H strains has indicated that some monoclonal antibodies share binding-site idiotypic determinants with TEPC 15, although these clones represent a minority of the precursor cells. In addition to providing insights into the heterogeneity and expression of the murine B-cell repertoire, these studies emphasize structural relationships between PC-specific clonotypes. Within the BALB/c strain, some antibodies share combining-site-related idiotypic specificities with TEPC 15, but differ in other variable region determinants. Among allotypically distinct strains, there exists a remarkable similarity of variable region determinants in at least a minority of antibodies.

Animals↗
PubMed1977

Idiotype-specific neonatal suppression of phosphorylcholine-responsive B cells.

The effect on neonatal anti-idiotypic suppression on the expression of B cells of the T15 clonotype has been investigated at the level of individual clonal precursor cells. The results indicate that B cells of the T15 clonotype are almost completely eliminated from the repertoire for four months after neonatal injection of allogeneic anti-idiotypic serum. The degree of this suppression is dependent on the amount of anti-idiotypic antibody administered and is less profound if anti-idiotypic antibody is given after the first week of life. No suppression was observed when anti-idiotypic antisera were administered to mice 30 days of age or older, which may indicate that immature B cells are the population most susceptible to suppression. However, since suppression could be reversed by administration of T15 myeloma protein several days after injection of anti-idiotype, the inability to suppress adult BALB/c mice may have been due to the high level of T15 idiotype normally present in their serum. Finally, phosphorylcholine-responsive B cells of identifiable clonotypes other than T15, even a clonotype sharing antigen-combining site determinants with T15, appear unaffected by anti-T15 suppression.

Aging↗
PubMed1977

Segregation at a locus determining an immunoglobulin genetic marker for the light chain variable region affects inheritance of expression of an idiotype.

Previous investigations have demonstrated close genetic linkage between loci governing expression of strain-specific idiotypes and immunoglobulin heavy (H) chain allotype (i.e., the C(H) locus). This linkage is presumed to reflect polymorphism of V(H) genes (or of their expression) linked to the polymorphic C(H) locus. That there was no apparent involvement of light (L) chain loci (thought to be unlinked to H chain) in inheritance of the idiotype-positive (Id(+)) phenotype was surprising, because the L chain is required for formation of each of the idiotypes studied at the chemical level. However, previous studies involving backcrosses of F(1)(Id(+) x Id(-)) mice to the Id(-) parental strain have never employed as the Id(-) parent one of several inbred strains shown by G. M. Edelman and P. D. Gottlieb [(1970) Proc. Natl. Acad. Sci. USA 67, 1191-1199] to express a V(L)-region polymorphism. Among backcrosses performed in the present study, one involved the A/J strain as the Id(+) parent and the PL/J strain, one of the several strains with and L chain polymorphism, as the Id(-) parent. Whereas in three other backcrosses performed, idiotype expression segregated with H chain allotype, in the backcross to PL/J, all mice producing the characteristic A/J Id(+) phenotype were A/J allotype(+), but not all A/J allotype(+) mice were Id(+). Typing of backcross progeny for the Ly-3 thymocyte alloantigens, controlled by a locus closely linked to that governing the L chain polymorphism (called the VK-1 locus), indicated that only the Ly-3 heterozygotes expressed the characteristic A/J Id(+) phenotype. Thus, all Id(+) backcross mice inherited both the H chain allotype and the Ly-3 locus (and closely linked L chain-related locus) from the Id(+) A/J strain. This suggests that, when strains with L chain polymorphisms are included in genetic studies of idiotype expression, segregation of loci governing L chain expression may be found to contribute to inheritance of the Id(+) character. In addition, these studies may be taken as further evidence that the V(L)-region repertoire of the PL/J strain may differ considerably from that of most other inbred strains of mice.

Animals↗
PubMed1977

Ia antigens on murine thymocytes: detection by cellular radioimmunoassay.

An indirect radioimmunoassay was used to detect Ia antigenic determinants at the cell surface of thymic and splenic cells. An A.TH anti-A.TL serum was used as a source of anti-Ia antibodies which could bind to H-2a or H-2k target cells. Bound alloantibody was then detected with 125I anti-mouse IgG1 antibody. This assay system revealed Ia antigens on thymic T cells which are not usually detectable by cytotoxicity assays, and has the potential for quantitative measurement of such determinants on cells. Critical adsorption studies were performed to confirm that Ia antigens were in fact the targets of the alloantiserum tested.

Animals↗
PubMed1976

Allogeneic carrier-specific enhancement of hapten-specific secondary B-cell responses.

We have analyzed the capacity of carrier-specific T cells to enhance the immune response of hapten-specific secondary B cells which do not share genes in the H-2 complex with the T cells. For this analysis we have used the in vitro splenic focus technique which allows assessment of monoclonal responses of B cells isolated in splenic fragment cultures of irradiated reconstituted carrier primed mice. A previous report from this laboratory demonstrated that syngeny in the I region of the H-2 complex was necessary between collaborating hapten-specific primary (nonimmune) B cells and carrier-specific T cells for responses yielding IgG1 but not IgM antibody. These findings lead up to postulate that the expression of I-region gene products on the surface of primary B cells and I-region syngeny with collaborating carrier-specific T cells were essential elements in the triggering events leading to IgG1 synthesis by primary B cells. The results presented in the present report indicate that, unlike primary B cells, the majority of secondary B cells can be stimulated to produce IgG1 antibody in carrier-primed allogeneic recipients. Although the enhancement of secondary IgG1 responses is slightly greater with syngeneic T cells, the allogeneic collaborative interaction requires both carrier priming of recipient mice and stimulation with the homologous hapten-carrier complex and thus appears to be specific. These findings clearly discriminate secondary from primary B cells and indicate that the mechanism of stimulation of secondary B cells to yield IgG1-producing clones differs fundamentally from the stimulation of primary B cells in that the requisite for I-region syngeny is obviated.

Animals↗
PubMed1976

Expression of Ia antigens on hapten-specific B cells. I. Delineation of B-cell subpopulations.

The nonimmune adult spleen contains at least two B-cell subpopulations. The majority of primary B cells express cell surface Ia determinants and have the capacity to give rise to IgG antibody-producing clones after T-cell dependent antigenic stimulation. There is also a small subpopulation of primary B cells which are, by definition, Ia negative, since their activity is not eliminated by negative selection with anti-Ia serum and complement. The Ia-negative B cells give rise to clones that produce only IgM antibody. These B-cell subsets may form a continuum in B-cell maturation, or they may exist as discrete B-cell lineages. Since the cellular expression of Ia antigens appears to correlate with the ability of the B cell to generate IgG-producing clones, it is speculated that Ia molecules may have a role in the IgM to IgG B-cell switch mechanism.

Animals↗
PubMed1976

In vitro tolerance induction of neonatal murine B cells.

The susceptibility of neonatal and adult B lymphocytes to tolerance induction was analyzed by a modification of the in vitro splenic focus technique. This technique permits stimulation of individual hapten-specific clonal precursor cells from both neonatal and adult donors. Neonatal or adult BALB/c spleen cells were adoptively transferred into irradiated, syngeneic, adult recipients which had been carrier-primed to hemocyanin (Hy), thus maximizing stimulation to the hapten 2,4-dinitrophenyl coupled by Hy (DNP-Hy). Cultures were initially treated with DNP on several heterologous (non-Hy) carriers and subsequently stimulated with DNP-Hy. Whereas the responsiveness of adult B cells was not diminished by pretreatment with any DNP conjugate, the majority of the neonatal B-cell response was abolished by in vitro culture with all of the DNP-protein conjugates. During the 1st wk of life, the ability to tolerize neonatal splenic B cells progressively decreased. Thus, tolerance in this system is: (a) restricted to B cells early in development; (b) established by both tolerogens and immunogens; (c) achieved at low (10(-9) M determinant) antigen concentrations; and (d) highly specific, discriminating between DNP- and TNP-specific B cells. We conclude that: (a) B lymphocytes, during their development, mature through a stage in which they are extremely susceptible to tolerogenesis; (b) the specific interaction of B-cell antigen receptors with multivalent antigens, while irrelevant to mature B cells, is tolerogenic to neonatal (immature) B cells unless antigen is concomitantly recognized by primed T cells; and (c) differences in the susceptibility of immature and mature B lymphocytes to tolerance induction suggest intrinsic differences between neonatal and adult B cells and may provide a physiologically relevant model for the study of tolerance to self-antigens.

Age Factors↗
PubMed1976

The acquisition of b-cell competence and diversity.

The in vitro splenic focus technique has been used to establish the characteristics of the repertoire of B cells in adult BALB/c mice, its stimulation and development. Evidence is presented that the technique yields an accurate estimate of the precursor cell frequency by providing monoclonal responses of a constant portion (4%) of transferred B cells. Since the monoclonally derived antibodies can be analyzed, it has also been possible to determine the isotype and clonotype of many of the resultant antibodies. The adult spleen can be shown to contain a vast repertoire (over 10(7) clonotypes) of primary B cells, each clonotype represented by from less than ten to as many as 10(4) responsive cells. Antigenic stimulation of these primary B cells is highly specific and apparently affinity dependent and leads to both antibody-forming cell clone formation and a population of secondary B cells. Secondary B cells, though derived from the same clonotypes, differ from primary B cell in several characteristics, including a lower degree of secificity of antigenic stimulation. In addition, secondary B cells can be stimulated in collaboration with allogeneic antigen-specific T cells to yield clones producing antibody of the IgG1 isotope, whereas primary B cells similarly stimulated produce only IgM antibodies. Analyses of neonatal B cells indicate that the acquisition of the adult repertoire is a highly patterned process. Thus, whereas 10(4) clonotypes are present at birth, more and more clonotypes are acquired in a highly ordered pattern during the first few weeks of life. It is concluded that this process is probably evolutionarily determined, since antigen-driven events appear to play no role. However, since developing B cells can be shown to be exquisitely sensitive to tolerance induction, negative antigenic selection may play an important role, particularly in eliminating clonotypes recognizing self-determinants for which the species is polymorphic.

Animals↗