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M C Nussenzweig

Publications and source records attributed to M C Nussenzweig.

16 recordsLinked to original sources
PubMed1992

Functional reconstitution of an immunoglobulin antigen receptor in T cells.

Humoral immune responses are initiated by binding of antigen to the immunoglobulins (Igs) on the plasma membrane of B lymphocytes. On the cell surface, Ig forms a complex with several other proteins, two of which, MB-1 and B29, have been implicated in receptor assembly. We have reconstituted Ig receptor function in T lymphocytes by transfection of cloned receptor components. We found that efficient transport of IgM to the surface of T cells required coexpression of B29. Furthermore, IgM and B29 alone were sufficient to reconstitute antigen-specific signal transduction by Ig in the transfected T cells. Crosslinking of IgM with either antireceptor antibodies or antigen induced a calcium flux, phosphoinositol turnover, and interleukin secretion in T cells. These experiments establish a requirement for B29 in Ig receptor function, and suggest that the signaling apparatus of T and B cells is structurally homologous.

Antibodies, Monoclonal↗
PubMed1992

Chromosomal position of rearranging gene segments influences allelic exclusion in transgenic mice.

Formation of a complete immunoglobulin heavy-chain transcription unit involves the ordered rearrangement of variable (V), diversity (D), and joining (J) region gene segments. In antibody-producing cells, this process is regulated such that only one of two antibody genes is expressed. Experiments with transgenic mice suggest that this mechanism, known as allelic exclusion, is mediated through the membrane-bound form of the immunoglobulin heavy chain. However, in all transgenic lines produced to date exclusion of the endogenous genes by the transgene is incomplete. To characterize the molecular basis for this escape from regulation, we have examined the rearrangements of endogenous immunoglobulin heavy-chain genes. We find that a transgene that encodes the membrane-bound form of human IgM efficiently inhibits rearrangements of endogenous gene segments located at the 5' end of the heavy-chain locus. However, recombining elements found at the 3' end of the locus escape and continue to undergo recombination. A transgene that encodes the secreted form of the same immunoglobulin protein has no effect on recombination, regardless of position of the recombining segment in the chromosome. These results have important implications for our understanding of the control of allelic exclusion.

Alleles↗
PubMed1991

Trans-splicing as a possible molecular mechanism for the multiple isotype expression of the immunoglobulin gene.

We analyzed the molecular mechanism for the immunoglobulin (Ig) multiple isotype expression using a transgenic mouse (TG.SA) model system. Though most of the endogenous mu chain expression was excluded by the expression of the human rearranged mu transgene in the TG.SA mouse, a significant portion of splenic B lymphocytes could express the transgenic human IgM and endogenous mouse IgG simultaneously after stimulation with lipopolysaccharide and interleukin 4. The fluorescence-activated cell sorter-purified population of the human IgM+/mouse IgG+ cells expressed mRNA that consisted of properly spliced sequences of the transgenic VHDJH and the endogenous mouse C gamma genes (trans-mRNA), together with the transgenic human mu mRNA and germline transcripts of the mouse C gamma gene, without apparent rearrangement of the transgene. We also found that a lymphoma tumor, derived from the cross between the TG.SA mouse and another transgenic mouse carrying Ig H chain enhancer-driven c-myc oncogene, expressed about equal levels of the trans-mRNA and the transgenic mu mRNA without DNA rearrangement in either the transgene or the endogenous mouse switch region. These findings strongly support our previous proposal that the trans-splicing can account for the multiple isotype expression in this transgenic model and also suggest that novel molecular mechanism(s) might be involved in this reaction.

Animals↗
PubMed1989

Mapping and characterization of an X-linked processed gene related to MYCL1.

A DNA sequence with homology to the myc family of proto-oncogenes has been characterized and found to be a processed gene related to L-MYC (MYCL1). This processed gene (MYCL2) was isolated by cross-hybridization to an oligonucleotide probe synthesized from the C-MYC (MYC) sequence in a highly conserved region of the myc gene family. Sequence analysis of MYCL2 revealed an open reading frame of 1194 bp with no intervening sequences and strong homology to the recently published DNA sequence of MYCL1. Southern and Northern blot analyses of DNAs and RNAs from small cell lung carcinomas confirmed its MYCL1 homology. Mapping of MYCL2 by somatic cell hybrids places this sequence on the long arm of the X chromosome in bands q22----q28.

Animals↗
PubMed1989

Immunoglobulin double-isotype expression by trans-mRNA in a human immunoglobulin transgenic mouse.

We have studied immunoglobulin double-isotype expression in a transgenic mouse (TG.SA) in which expression of the endogenous immunoglobulin heavy chain locus is almost completely excluded by a nonallelic rearranged human mu transgene. By flow-cytometric analyses, we have shown that a small, but significant, portion (about 4%) of transgenic spleen cells expresses human mu (transgene) and mouse gamma (endogenous) chains when cultured in vitro with bacterial lipopolysaccharide and interleukin 4. By using amplification of cDNA by the polymerase chain reaction, followed by cloning and sequencing of the amplified cDNA fragment, we have demonstrated expression of trans-mRNA consisting of the transgenic variable and endogenous constant (gamma 1) region sequences. Such trans-mRNA could be produced by either switch recombination or trans-splicing between the transgene and endogenous sterile gamma 1-gene transcripts. These results indicate that trans-splicing might be a possible mechanism for the immunoglobulin double-isotype expression in normal B lymphocytes that have not rearranged the second expressed constant region gene.

Animals↗
PubMed1988

A human immunoglobulin gene reduces the incidence of lymphomas in c-Myc-bearing transgenic mice.

Transgenic mice carrying an immunoglobulin enhancer-driven c-myc oncogene develop rapid-onset pre-B cell lymphomas. The incidence of these malignancies is greatly reduced when an additional transgene encoding the membrane-bound form (but not the secreted form) of human Ig mu is bred into the susceptible strain. This suppressive effect correlates with a subtle alteration in B-cell development induced by the immunoglobulin transgene.

Abelson murine leukemia virus↗
PubMed1988

Allelic exclusion in transgenic mice carrying mutant human IgM genes.

Expression of the membrane-bound version of the human mu chain in transgenic mice results in the allelic exclusion of endogenous mouse Ig heavy chain genes (6). The secreted version of the human Ig transgene has no such effect. F1 hybrid animals that carry transgenes for both secreted and membrane-bound human mu chains produce both forms of the human heavy chain while strongly suppressing endogenous mouse mu expression. The simultaneous expression of the two rearranged transgenes in primary B cells suggests that allelic exclusion operates before the formation of a second functionally rearranged heavy chain gene in vivo.

Alleles↗
PubMed1987

Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin mu.

Antibody-producing cells display a special form of regulation whereby each cell produces immunoglobulin from only one of its two sets of antibody genes. This phenomenon, called allelic exclusion, is thought to be mediated by the product of one heavy chain allele restricting the expression of the other. Heavy chains are synthesized in two molecular forms, secreted and membrane bound. In order to determine whether it is specifically the membrane-bound form of the immunoglobulin M (IgM) heavy chain (mu) that mediates this regulation, transgenic mice were created that carry a human mu chain gene altered so that it can only direct the synthesis of the membrane-bound protein. The membrane-bound form of the human mu chain was made by most of the B cells in these animals as measured by assays of messenger RNA and surface immunoglobulins. Further, the many B cells that express the human gene do not express endogenous mouse IgM, and the few B cells that express endogenous mouse mu do not express the transgene. Thus, the membrane-bound form of the mu chain is sufficient to mediate allelic exclusion. In addition, the molecular structures recognized for this purpose are conserved between human and mouse systems.

Alleles↗
PubMed1983

Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice.

Clone 33D1 is a mouse-rat hybridoma that secretes a specific anti-dendritic cell (DC) monoclonal antibody (14). Because the antibody kills DC in the presence of rabbit complement, it can be used to study the functional consequences of selective DC depletion. Previous data on the cell specificity of 33D1 were first extended. By cytotoxicity (rabbit complement) and indirect immunofluorescence (biotin-avidin technique), 33D1 reacted with DC but not with macrophages nor other splenocytes. In contrast, the monoclonal antibody, F4/80 (15), reacted with macrophages but not DC. The functional assay evaluated in this paper was stimulation of the primary mixed leukocyte reaction (MLR). 33D1 antibody itself did not inhibit stimulation by enriched populations of DC. In the presence of complement, 33D1 killed DC and ablated stimulatory function. The effect of 33D1 and complement on MLR stimulation by heterogenous cell mixtures was then evaluated. Removal of DC from unfractionated spleen suspensions reduced stimulatory capacity 75-90 percent, comparable to that produced with specific anti-Ia antibody and complement. Stimulation of both proliferative and cytotoxic responses was reduced. DC depletion had similar effects on MLR generated across full strain differences, or across selected subregions (H2I, H-2K/D) of the major histocompatibility complex. To further compare the functional properties of spleen DC and macrophages, MLR stimulation by adherent and nonadherent fractions of spleen were tested separately. 62 +/- 8 percent of the total stimulatory capacity of spleen was in the plastic adherent population. Activity was ablated greater than 90 percent after elimination of DC. MLR stimulation by 24-h cultures of spleen adherent cells, which contained a three- to sixfold excess of Ia(+) macrophages, was also ablated when DC were removed. Stimulation by nonadherent spleen was more resistant, but was reduced 50-75 percent by 33D1 and complement. The function of spleen cells treated with 33D1 or anti-Ia antibody and complement was restored with a small inoculum of purified DC. The latter corresponded to 0.5 percent of total stimulator cells and were enriched by previously described techniques that did not require the 33D1 antibody. We conclude that the DC, a trace component of mouse spleen, is the principal cell type required for stimulation of the primary MLR. Because other cells are not immunogenic, but do express Ia and H-2 alloantigens, DC likely represent the critical accessory cell required for the induction of lymphocyte responses.

Animals↗
PubMed1982

A monoclonal antibody specific for mouse dendritic cells.

Dendritic cells (DC) are a small subpopulation of lymphoid cells with distinctive cytologic features, surface properties, and functions. This report describes the DC-specific antibody (Ab) secreted by clone 33DI. Rat spleen cells immune to mouse DC were fused to the P3U myeloma. Hybrid culture supernatants were screened simultaneously against DC, a macrophage (M phi) cell line, and mitogen-stimulated lymphoblasts. 33DI Ab specifically killed 80-90% of DC from spleen and lymph node, but no other leukocytes, including Ia+ and Ia- M phi (Ia, I-region-associated antigen,). Quantitative binding studies with 5H-labeled 33D1 Ab showed that DC had an average of 14,000 binding sites per cell. Binding to DC was inhibited with Fab fragment of 33D1 Ab but not with a panel of other monoclonal antibodies, including anti-Ia Ab. Adherence and flotation procedures that enriched for DC enriched for 3H-labeled 33D1 Ab binding in parallel. 33D1 antigen was not detectable on: M phi from spleen, peritoneal cavity, and blood; three M phi cell lines; lymphocytes; granulocytes; platelets; and erythroid cells. DC continued to express the 33D1 antigen after 4 days in culture, whereas M phi and lymphocytes did not acquire it. Quantitative and autoradiographic studies confirmed that spleen and lymph node suspensions contain less than 1% DC. We conclude that 33D1 Ab detects a stable and specific DC antigen and can be used to monitor DC content in complex lymphoid mixtures.

Animals↗
PubMed1980

Dendritic cells are accessory cells for the development of anti-trinitrophenyl cytotoxic T lymphocytes.

This study establishes that dendritic cells (DC) are the critical accessory cells for the development of anti-trinitrophenol (TNP) cytotoxic T lymphocytes (CTL) in vitro. We developed a model in which nylon wool-nonadherent spleen cells were used both as the responding and stimulating cells, the latter having been TNP-modified and x-irradiated. Thy-1-bearing CTL developed in C57BL/6, B6D2F1, and CBA mice only when small numbers of DC were added. Maximal responses in 5-d cultures were achieved with 0.5-1 DC/100 responding T cells. The DC did not have to be TNP modified directly. Anti-Ia and complement inactivated accessory cells, whereas similar treatment of the responders had no effect. DC exposed to ultraviolet radiation were ineffective, but x-irradiated DC were fully active. Culture media from DC, or from DC-nylon wool-passed spleen T cell cocultures that contained abundant CTL, would not substitute for viable DC. Enriched preparations of macrophages (M phi) were obtained from blood, peritoneal cavity, and spleens of BCG-immune and unprimed mice. M phi added at doses of 0.2-4% were weak or inactive as accessory cells. The level of Ia antigens on test M phi populations was quantitated and visualized by binding of a radioiodinated monoclonal anti-I-Ab,d antibody, clone B-21. M phi that bore substantial amounts of Ia from all organs were weak accessory cells. Addition of M phi to DC-T cell cocultures produced inhibitory effects, usually at a dose of 2% M phi. In contrast, 0.5% Ia-bearing M phi from BCG-immune boosted mice inhibited > 80% of the DC-mediated CTL response. Addition of indomethacin reversed M phi inhibition, and 10(-9) M prostaglandin E2 in turn blocked the indomethacin effect. Indomethacin also restored a low level of accessory cell function in immune-boosted adherent peritoneal cells, but not in preparations of monocytes and spleen M phi. Small numbers of DC were identified in preparations of immune-boosted peritoneal cells and may have accounted for the observed accessory activity. We conclude that the development of anti-TNP CTL is an immune response in which (a) DC are the critical accessory cells; (b) Ia-bearing M phi are weak or inactive; and (c) M phi can inhibit DC-mediated response by an indomethacin-sensitive mechanism.

Animals↗
PubMed1980

Contribution of dendritic cells to stimulation of the murine syngeneic mixed leukocyte reaction.

We have studied the proliferative response of unprimed T cells to syngeneic dendritic cells (DC) (syngeneic mixed leukocyte reaction [SMLR]) in cultures of mouse spleen and lymph node. T cells purified by passage over nylon wool contain few DC and exhibits little proliferative activity during several days of culture. Addition of small numbers of purified syngeneic DC induces substantial, dose-dependent, T cell-proliferative responses that peak at day 4-5. B cells purified on anti-Ig-coated plates do not respond to DC at all doses tested. DC culture medium does not induce proliferation, and coculture of DC and T cells is required. Purified mouse B and T lymphocytes stimulate SMLR weakly if at all. Likewise, peritoneal and spleen macrophages are weak or inactive. Therefore, DC are potent and possibly unique primary cells for stimulating the SMLR in mice. sIg- spleen and lymph node cells show extensive background proliferative responses in vitro, and fail to respond to small numbers of purified DC. If the sIg- cells are treated with anti-Ia and complement, or passed over nylon wool, DC are removed and proliferative activity falls. Proliferative activity is restored by adding back DC at levels similar to those present in sIg- cells (1-2%). Thus, DC-dependent, T cell proliferation probably occurs in all spleen and lymph node cultures. As expected from previous work (6), DC are also potent inducers of allogeneic MLR. On a per DC basis, the syngeneic response is 10 times weaker than the allogeneic MLR, and it is not accompanied by the development of cytotoxic lymphocytes. The magnitude of the SMLR was not altered by antigen priming, and DC maintained in isologous rather than fetal calf serum were active stimulators. Therefore, syngeneic stimulation appears to be an intrinsic property of DC, and modification by exogenous agents does not seem to be required. Coculture of DC and T cells results in the development of cell clusters that can be isolated and characterized directly. The clusters account for 10-20% of the viable cells in the culture, but contain greater than 80% of the responding T cells and stimulating DC by morphologic and surface-marker criteria. The efficient physical association of DC and responding T cells implies specific cell-cell recognition. We conclude that the SMLR reflects the ability of T cells, or some subpopulation of T cells, to interact with and proliferate in response to small numbers of DC.

Animals↗