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N R Klinman

Publications and source records attributed to N R Klinman.

At least 55 records · Page 3Linked to original sources
PubMed1986

Surface IgG-bearing cells retain the capacity to secrete IgM.

Our previous studies indicated that a large proportion of surface IgG+-primed B cells give rise to clones secreting IgM antibodies. Due to the implications of this result relative to molecular mechanisms of class switching, it was important to document that the surface IgG had been endogenously synthesized by the surface IgG+ cells and was not present as a result of cytophilic IgG. Therefore, spleen cells from immunized mice were treated sequentially with anti-immunoglobulin and protease which removed greater than 99% of surface immunoglobulin. After overnight incubation to allow resynthesis of surface immunoglobulin, the treated cells were sorted for surface IgG-bearing cells and were transferred to carrier-primed, irradiated adoptive recipients for analysis in the splenic focus assay. It was found that the majority of antibody-secreting clones derived from these surface IgG+ B cells still synthesized IgM. These data are discussed relative to current concepts of molecular mechanisms of immunoglobulin class switching.

Animals↗
PubMed1986

The frequency and fine specificity of B cells responsive to (4-hydroxy-3-nitrophenyl)acetyl in aged mice.

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.

Aging↗
PubMed1986

Preferential expression of variable region heavy chain gene segments by predominant 2,4-dinitrophenyl-specific BALB/c neonatal antibody clonotypes.

The B-cell repertoire in neonatal mice contains predominant clonotypes that are reproducibly expressed at particular times after birth. We have isolated and sequenced heavy and light chain cDNA clones from three 2,4-dinitrophenyl-specific neonatal hybridomas. Two of these hybridomas (TF2-36 and TF5-139) express idiotypes (Ids) that predominate during the first days after birth, and the third hybridoma (TF2-76) expresses an Id that predominates during the second week after birth. The heavy (H) chain variable (V) region of the TF2-76 hybridoma protein is encoded by a member of the 7183 VH family, one of eight families of murine VH genes that have been defined by Brodeur and Riblet [Brodeur, P. H. & Riblet, R. (1984) Eur. J. Immunol. 14, 922-930]. Members of this family have been found to undergo a disproportionately high frequency of rearrangement in fetal and neonatal liver pre-B-cells. Because the 7183 VH family is located close to the H chain joining (J) region gene segments, JH, other workers have proposed that VH rearrangement frequency is related to distance from the JH segments. However, the two earlier-appearing predominant clonotypes expressed by TF2-36 and TF5-139 hybridoma proteins utilize a member of the 36-60 VH family, probably VH 1210.7, which is located distal to the JH gene segments on chromosome 12. Since 20-30% of day 3 dinitrophenyl-specific B cells express either the Id(TF2-36) or the Id(TF5-139), the VH 1210.7 gene must be utilized at high frequency early in development. These results indicate that the utilization of rearranged VH segments is strongly influenced by factors other than distance from JH.

Animals↗
PubMed1985

Strain-specific silencing of a predominant antidextran clonotype family.

The immune response to dextran is characterized by marked phenotypic differences among murine strains. In particular, Igha strains, as opposed to strains of other Igh haplotypes, respond relatively vigorously to dextran B1355 fraction S (DEX), producing predominantly antibodies bearing the lambda light chain, and specific for the alpha(1----3) glucose linkage. We have investigated this disparity in BALB/c (Igha) vs. C.B20 (Ighb) mice at the individual precursor cell level. Consistent with previous findings (7-9, 35, 40, 42, 43), there was a 10-fold higher frequency of lambda-bearing splenic B cells specific for the alpha(1----3) linkage in Igha mice. As with previously studied (25-27) predominant specificities, the origin of this high frequency of lambda-bearing alpha(1----3) DEX-specific B cells appears to be a reflection of a high expression of this specificity in surface Ig (sIg)-negative cells emerging from the bone marrow generative cell pool. Surprisingly, although C.B20 mice (Ighb) have a low frequency of lambda-bearing alpha(1----3) DEX-specific B cells in their mature primary splenic population, the frequency of precursor cells of this clonotype in their sIg- bone marrow cell population is equivalent to that of BALB/c sIg- cells. These cells could only be stimulated in allotype allogeneic (Igha), as opposed to allotype syngeneic (Ighb), carrier-primed irradiated recipients. This finding was confirmed by the finding that a high proportion of antidextran hybridoma cell lines derived from C.B20 bone marrow cells produced lambda-bearing alpha(1----3) DEX-specific antibodies that were IdX+. These findings have led us to conclude that the well-established phenotypic difference between Igha and Ighb mice with respect to the expression of lambda-bearing alpha(1----3) DEX-specific antibody responses is not, as previously assumed, the result of an inability of Ighb mice to generate B cells of this clonotype, but rather, is the product of environmental, possibly antiidiotypic, silencing of cells of this clonotype as they mature in Ighb mice.

Animals↗
PubMed1985

Analysis of an evolutionarily conserved antigenic site on mammalian cytochrome c using synthetic peptides.

Two synthetic peptides inclusive of the NH2-terminal N-acetyl-Gly-Asp-Val-Glu tetrapeptide of mammalian cytochrome c (cyt c) were used in this study to address the question of whether mammals can respond immunologically to an evolutionarily conserved region of a protein. These peptides were assessed for their capacity (i) to act as immunogens for the production of anti-self cyt c antisera and (ii) to bind rabbit anti-rodent cyt c antibody. The findings from these studies indicate the existence of an immunogenic determinant in an evolutionarily conserved region of cyt c that contains residues 1-4. This determinant can induce anti-self cyt c antibodies whether presented as a peptide on a carrier protein or in the context of the intact molecule as polymerized mammalian cyt c.

Amino Acid Sequence↗
PubMed1985

Tolerance and B cell repertoire establishment.

We have probed the mechanism by which immature B cells are uniquely susceptible to antigen-induced inactivation. Our studies have demonstrated that this tolerance trigger is an active process that requires both energy metabolism and the biosynthesis of various macromolecules, including protein, RNA, and DNA. However, the tolerance trigger is resistant to inhibitors of patching and capping, as well as an inhibitor of mitosis. The tolerance trigger requires a high-affinity interaction between a multivalent antigen and the cells' Ig receptor, but apparently does not require interactions with other cell surface molecules, or interactions with T cells or macrophages. Our efforts to demonstrate the physiological applicability of this tolerance trigger have concentrated on an attempt to demonstrate potentially self-reactive cells within the immature bone marrow population that do not appear in the mature splenic B cell population. To date we have identified prereceptor B cells of several specificities whose frequency is much lower in the spleen and whose elimination appears to involve tolerance rather than antiidiotypic regulation. However, the demonstration that such cells are eliminated by contact with self-antigens has not as yet been accomplished.

2,4-Dinitrophenol↗
PubMed1985

Differences in antibody repertoires for (4-hydroxy-3-nitrophenyl)acetyl (NP) in splenic vs immature bone marrow precursor cells.

To evaluate the contribution of environmental regulatory mechanisms in fashioning the primary B cell repertoire, we have compared the repertoire of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific primary splenic B cells with that of precursor cells present as surface immunoglobulin-negative (sIg-) cells in adult bone marrow of C.B20 (Ighb) mice. Previous analyses using a variety of antigens have led to the conclusion that the antibody repertoire expressed in the spleen is similar to that expressed in newly generated B cell precursors with respect to both repertoire diversity and the representation of various predominant clonotypes. However, in the response to NP of C.B20 precursor cells, two marked disparities have been identified between the repertoire of sIg- bone marrow cells vs splenic precursor cells. The first concerns precursor cells that give rise to lambda-bearing NP-specific antibodies with heteroclitic fine specificity. Such antibodies normally dominate the primary response of Ighb mice; however, the representation of precursor cells giving rise to lambda-bearing antibodies is disproportionately low in the sIg- bone marrow cell population of C.B20 mice. Thus, during the maturation of these cells, post-sIg receptor expression, there is an apparent increase in the proportionate representation of lambda-bearing NP-specific cells. The second disparity concerns precursor cells whose antibody products bear kappa-light chains and exhibit high affinity and homoclitic binding for the NP haptenic determinant. Such precursor cells are poorly represented in the spleen, but represent a sizeable proportion of the sIg- NP-specific precursor cell population. Thus, there seems to be a selective elimination of high affinity, kappa-homoclitic anti-NP antibody-bearing cells as they acquire their sIg receptors. The elimination of this cell population could partially account for the dominance of lambda-heteroclitic antibodies in the serum responses to NP of C.B20 mice.

Animals↗
PubMed1984

Interaction of gangliosides with B cells in splenic fragment cultures.

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.

Animals↗
PubMed1984

B cell repertoire diversity to PR8 influenza virus does not decrease with age.

The affect of aging on the B cell repertoire was investigated at the clonal level by studying the response of 24- to 26-mo-old BALB/c mice to the PR8 influenza virus (H1N1). By using the splenic fragment culture technique, it was found that the frequency of B cells specific for either the intact virion or the HA does not change with age. The anti-HA monoclonal antibodies obtained from culture were additionally analyzed for fine specificity by binding in RIA to a panel of six H1 variant viruses. This analysis showed a considerable similarity in the distribution and diversity of reactivity patterns between monoclonal antibodies derived from splenic B cells of young vs aged mice. These findings indicate that repertoire expression per se may not be truncated in aged mice, and imply that reductions in the diversity of serum antibodies in aged mice may be due to environmental regulatory mechanisms.

Aging↗
PubMed1984

Membrane and metabolic requirements for tolerance induction of neonatal B cells.

The metabolic requirements of tolerance induction of immature B cells has been analyzed through the use of various putative inhibitors. The study utilizes the splenic fragment assay in which tolerance induction of individual B cells can be examined. Concentrations of inhibitors were determined which, if removed after the first 18 hr of culture, before antigenic stimulation, had no inhibitory effects. Thus, by adding tolerogen in the presence or absence of inhibitor during the first 18 hr of culture, the effect of that inhibitor on tolerance could be assessed. By using this protocol, the data indicate that several metabolic functions of the cell are necessary for tolerance induction to occur, including RNA biosynthesis, DNA biosynthesis, and a methyltransferase reaction, because drugs that interfere with these metabolic processes also prevent tolerance induction. Our previous studies indicated that protein biosynthesis and energy generation are also required. However, drugs that interact with the cytoskeletal structure of the cell and inhibit surface immunoglobulin capping do not interfere with tolerance induction. Moreover, colchicine, which inhibits cell division, does not inhibit B cell tolerance. Collectively, the results provide compelling evidence that the mechanism of immature B cell tolerance involves an active process requiring several metabolic activities of the cell.

Animals↗
PubMed1983

Role of variable region gene expression and environmental selection in determining the antiphosphorylcholine B cell repertoire.

To evaluate the role of environmental selective processes, as opposed to variable region gene expression, in the determination of B cell repertoire expression, we have assessed the phosphorylcholine (PC)-specific repertoire of precursor cells that remain in bone marrow cell populations after the removal of surface immunoglobulin (sIg)-bearing cells. Such cells are assumed to represent a stage in B cell maturation before the expression of sIg, and thus at a time when they have not as yet interfaced with environmental influences that operate through sIg receptors such as antigenic stimulation, tolerance, or antiidiotypic regulation. The repertoire as expressed in these cells, therefore, should reflect the readout of immunoglobulin variable region genes as they are expressed in progenitors to B cells. The results of these studies indicate that, as in mature primary B cell pools of BALB/c mice, the majority of PC-responsive sIg- bone marrow cells are of the T15 clonotype. Thus, environmental selective mechanisms would not appear to be required for the high frequency of B cells of the T15 idiotype in the primary B cell repertoire of BALB/c mice. Analysis of the sIg- bone marrow cells in (CBA/N X BALB/c)F1 male mice demonstrated that the deficit of PC-responsive mature B cells, which is a characteristic of this murine strain, must occur after receptor expression, since a normal frequency of PC-responsive and T15-expressing cells is present in their sIg- bone marrow population. Finally, these same mice were used to obtain bone marrow cell preparations from individual leg bones, so as to permit an analysis of the occurrence of T15+ and T15- clonotypes within individual bone marrow populations. The findings from these studies indicate that T15+ B cells occur as a high frequency event within bone marrow generative cell pools. Furthermore, bone marrow populations that are positive for PC-responsive precursor cells often display multiple copies of such precursor cells that are exclusively either T15+ or T15-. This finding indicates that clonal expansion of cells within the B cell lineage apparently occurs before immunoglobulin receptor acquisition.

Animals↗
PubMed1983

B cell repertoire diversification precedes immunoglobulin receptor expression.

68 monoclonal antibodies specific for the hemagglutinin (HA) of the influenza virus, PR8, were obtained from sIg- bone marrow B cell precursors stimulated in splenic fragment cultures. Reactivity pattern (RP) analysis demonstrated that these anti-HA antibody responses included at least 29 distinguishable clonotypes. Comparison of the specificities of anti-HA antibodies obtained from sIg- bone marrow cells with those obtained from adult spleen cells indicates that the anti-HA repertoires of the two populations are comparable in diversity. Since the sIg- bone marrow B cell precursor pool presumably has not encountered V region-specific regulatory mechanisms in vivo, our data suggest that substantial diversification of the B cell repertoire precedes surface immunoglobulin (sIg) expression and subsequent interaction with environmental regulatory processes.

Animals↗
PubMed1983

Genetic and temporal control of neonatal antibody expression.

Two hybridoma cell lines were established with B cells derived from neonatal BALB/c spleen cells. The anti-dinitrophenyl (DNP) antibodies derived from these lines were characterized with respect to their isotype, affinity, and isoelectric point. Antiidiotypic reagents were prepared that permit an analysis of the representation of antibodies sharing idiotype with these two hybridomas in the developing and mature B cell pool of BALB/c mice (Igha) and other murine strains. One of the two antibodies, TF2-36, was found to be indistinguishable from 14% of anti-DNP monoclonal antibodies derived in fragment culture from spleen cells of 1-4-d-old BALB/c donors. B cells expressing this idiotype were found to represent approximately 2% of the anti-DNP-specific repertoire after the 1st wk of neonatal development and into adulthood. The second hybridoma antibody, TF2-76, was found to be expressed at very low levels during the first several days of neonatal development; however, B cells expressing this idiotype increased in frequency during the 2nd wk of neonatal development representing 7% of all DNP-responsive B cells 12-13 d after birth. The proportion of B cells expressing this idiotype also decreased to approximately 2% in adults. The relatively late appearance of B cells bearing this idiotype was confirmed by their susceptibility to tolerance induction after the 1st wk of neonatal development. Both the early neonatal clonotype, TF2-36, and the late neonatal antibody clonotype, TF2-76, were found to be expressed in a similar fashion in F1 mice constructed between Igha and Ighb parentals, but both were expressed at very low levels during the development of Ighb mice. Thus, the control of the magnitude of expression of these neonatal clonotypes appears to be associated with the Igh locus.

Aging↗
PubMed1983

Antigen responsiveness of the mature and generative B cell populations of aged mice.

The deficit of humoral immune responsiveness associated with aging was investigated at the level of individual antigen-specific B cells. It was found that mature dinitrophenyl (DNP)-responsive B cells isolated from the spleen of aged mice gave rise to clones of antibody-forming cells that were normal with respect to both the amount and relative affinity of anti-DNP antibody produced. However, although the proportion of immunoglobulin-bearing cells in the spleen of aged mice was normal, the proportion of cells that responded to T cell dependent DNP-specific stimulation (1.1 per 10(6) injected cells) was significantly lower than the proportion that responded when cells were obtained from the spleen of young mice (2.3 per 10(6) injected cells). To examine the origin of this diminution in antigen-responsive B cells, the responsiveness of precursor cells from the B cell generative pool isolated as the surface immunoglobulin negative (sIg-) cells within the bone marrow was evaluated. The frequency of DNP-responsive cells in both intact bone marrow cell suspensions and the sIg- subpopulation was not significantly different when such cells were isolated from aged vs. young individuals. Thus, it would appear that among the immunologic deficits associated with aging is a decrease in the proportion of antigen-responsive B cells, which is associated with maturation of B cell clones in the aged environment and occurs during the migration of cells from the bone marrow to the spleen.

Aging↗