Mu Heavy Chain Research Articles

Heparin alters the expression of different forms of immunoglobulin μ heavy chains and their associated proteins by pre-B cell lines and normal Ly-1 (CD5+) B cells

Studies presented here show that heparin alters immunoglobulin expression by murine pre-B cell lines and normal Ly-1 (CD5+) B cells. Previous studies have shown that pre-B cell lines 70Z/3 and NFS-5.3 express mu heavy chains in the cytoplasm and a small amount on the cell surface. Both these cytoplasmic and surface mu are disulfide-linked to omega (lambda 5) surrogate light chains and are noncovalently associated with iota (Vpre-B) variable region-like proteins. We show that culturing 70Z/3 with heparin reduces the amount of the membrane-form mu (micron) on the cell surface. Culturing NFS-5.3 with heparin similarly decreases the membrane-form mu; however, it increases the surface level of a pentameric mu molecule containing secreted-form mu (microS) heavy chains, disulfide-linked omega (lambda 5) chains, and noncovalently associated proteins. Culturing peritoneal B cells with heparin also increases the production of the secreted-form microS, detectable in this case by the secretion of classical pentameric IgM. Similarly, injecting heparin intraperitoneally increases IgM secretion by peritoneal Ly-1 B cells. Thus heparin could influence pre-B cell and B cell differentiation and function.

MRNA transcripts initiating within the human immunoglobulin mu heavy chain enhancer region contain a non-translatable exon and are extremely heterogeneous at the 5′ end

Transcription events are thought to precede gene rearrangement in the immunoglobulin (Ig) loci and may be the mechanism by which the various gene regions are made accessible for recombination. If this is the case, identification and characterization of transcripts from the Ig loci should permit a better understanding of the gene rearrangement process. We have isolated a 2.3 kb cDNA clone from the human pre-B cell line Nalm-1 that contains enhancer-specific sequences from the Ig heavy (H) chain gene locus. The 2.3 kb transcript initiated within the enhancer region and showed extreme 5' heterogeneity, with more than 50 initiation sites mapping near the Ig-specific octamer ATTTGCGT. Sequencing of the cDNA clone demonstrated that 644 nucleotides from the Ig enhancer region were incorporated as a leader exon spliced to the mu constant (Cmu) region. This leader exon contained many translation termination codons and may function to inhibit the translation of sterile Cmu polypeptides. Using an enhancer-derived probe, we detected two low-abundancy mRNA transcripts with sizes of 2.3 and 12 kb. Northern blot analysis suggested that the 12 kb transcript was the unspliced precursor mRNA of a VDJ rearrangement. The potential role of these enhancer-containing transcripts in the opening of the IgH chain gene for rearrangement and for class switching is discussed.

Detection of gene targeting by co-conversion of a single nucleotide change during replacement recombination at the immunoglobulin μ hesvy chain locus

A method is described for detecting targeted events at the mu heavy chain gene which relies on co-conversion (or co-exchange) of a point mutation with a selectable marker contained on a replacement vector. The vector, designed for application to IgM producing hybridomas, contains a single nucleotide change within the region of homology with the target gene which encodes a different allotypic determinant of IgM. In a model system where homologous recombination corrected a defective mu gene, the length of homology between this nucleotide change and the position of the double strand break in the vector was found to have a critical influence on the co-conversion frequency. In the vector design ultimately used for targeting in hybridomas, one in 1000-2000 stable transformants produced IgM with the allotype encoded by the exogenous DNA, and Southern blot analysis confirmed that these were derived by targeted integration. The sensitivity of the screening procedure using a monoclonal antibody specific to this allotype enabled a targeted clone to be detected in a pool of stable transformants when present at a frequency at least as low as one per cent. Several different modifications of the target locus were obtained as a consequence of alternative crossover positions and, in some cases, vector DNA concatenation.

Endogenous immunoglobulin expression in mu transgenic mice

Transgenic mice (M54) containing a functional mu heavy chain were examined to determine the effects of the transgene on rearrangement and expression of endogenous immunoglobulin genes. Two major novel findings are presented. (i) In transgenic mice, the expressed endogenous VH repertoire in LPS-generated B cell blasts and hybridomas is skewed toward expression of JH-proximal VH families (VH7183 and Q52). (ii) There is an increase in the frequency of B cells expressing lambda light chain genes in transgenic mice. Furthermore, in Abelson-MuLV transformed pre-B cells, VH to DJH is inhibited more than the D to JH rearrangement. The results presented indicate that the transgene skews the expressed VH repertoire by inhibiting the VH to DJH rearrangement while permitting an expansion of B cells expressing limited VH and lambda light chain genes.

A Mouse Monoclonal Antibody Reactive Preferentially with Human IgM λ

In analyzing mouse monoclonal antibodies (mAb) against a human IgM kappa paraprotein, we found an unusual mAb (LP4; gamma 2b kappa isotype) that reacted in an enzyme linked immunosorbent assay with all 5 IgM lambda but not with 8 IgM kappa or other myelomas. Neither isolated mu heavy nor lambda light chains were reactive with LP4 mAb. By immunofluorescence, LP4 mAb identified approximately 30% of IgM+ B cells and approximately 40% of mitogen-stimulated, IgM+ plasma cells from 4-7 normal blood samples. All LP4+ cells were IgM+. Biosynthetic analysis of the plasma cells revealed that LP4 mAb recognized most IgM lambda and a very minor proportion of IgM kappa molecules. This mAb provides a useful marker for the analysis of pre-B and B cell differentiation.

Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents

There are several demonstrations that misfolded or unassembled proteins are not transported along the secretory pathway, but are retained intracellularly, generally in the endoplasmic reticulum. For instance, B lymphocytes synthesize but do not secrete IgM, and only the polymeric form of IgM is secreted by plasma cells. The C-terminal cysteine of the mu heavy chain of secreted IgM (residue 575) is involved in the intracellular retention of unpolymerized IgM subunits. Here we report that the addition of reducing agents to the culture medium, at concentrations which do not affect cell viability, terminal glycosylation, or retention of proteins in the endoplasmic reticulum through the KDEL mechanism, induces secretion of IgM assembly intermediates by both B and plasma cells. Free joining (J) chains, which are not normally secreted by plasma cells unless as part of IgM or IgA, are also secreted in the presence of reducing agents. We propose a role for free thiol groups in preventing the unhindered transport of proteins through the secretory pathway. Under the scheme, assembly intermediates interact through their thiol groups between themselves and/or with unknown proteins of the endoplasmic reticulum. Such interactions may be prevented by altering the intracellular redox potential or by site-directed mutagenesis of the relevant cysteine residue(s).

The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain.

The murine pre-B cell-specific genes VpreB and lambda 5, as well as the murine gene for mu heavy chain, were introduced into Ltk- fibroblast cells which normally do not express these genes. Stable transfectants carrying these genes produced the corresponding proteins of 15.5, 21.5, and 75 kD. They secreted the three proteins as a triple complex that could be immunoprecipitated by mu heavy chain-specific antibodies, consisting of one VpreB, one lambda 5, and one mu heavy chain. The mu heavy chain and lambda 5 were disulfide-bonded with each other, while the VpreB protein was noncovalently associated. These experiments proved that the VpreB, lambda 5 and mu H chain proteins can form a heavy/light chain-like heterocomplex.

Complex Regulation of the Immunoglobulin μ Heavy-Chain Gene Enhancer: μB, a New Determinant of Enhancer Function

The B-lymphocyte-specific activity of the immunoglobulin mu heavy-chain gene enhancer has been attributed to the octamer motif (ATTTGCAT) present within the enhancer that binds a B-cell-specific factor designated NF-A2/OTF-2. However, significant residual enhancer activity even after deletion of this element has suggested the presence of a second critical functional determinant. We have used deletion and mutational analyses to define an element, microB (TTTGGGGAA), that is essential for B-cell-specific enhancer activity in S194 myeloma cells in the absence of the octamer. Transfection analysis in a panel of lymphoid cell lines suggests that the presence of either microB or octamer leads to considerable enhancer activity in cell lines representing later stages of B-cell differentiation, whereas both elements are needed for function in cell lines representing earlier stages. Furthermore, in contrast to the results in pre-B-cell lines, both microB and octamer elements function independently in certain T-cell lines in which the mu enhancer is active.

Complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microB, a new determinant of enhancer function.

The B-lymphocyte-specific activity of the immunoglobulin mu heavy-chain gene enhancer has been attributed to the octamer motif (ATTTGCAT) present within the enhancer that binds a B-cell-specific factor designated NF-A2/OTF-2. However, significant residual enhancer activity even after deletion of this element has suggested the presence of a second critical functional determinant. We have used deletion and mutational analyses to define an element, microB (TTTGGGGAA), that is essential for B-cell-specific enhancer activity in S194 myeloma cells in the absence of the octamer. Transfection analysis in a panel of lymphoid cell lines suggests that the presence of either microB or octamer leads to considerable enhancer activity in cell lines representing later stages of B-cell differentiation, whereas both elements are needed for function in cell lines representing earlier stages. Furthermore, in contrast to the results in pre-B-cell lines, both microB and octamer elements function independently in certain T-cell lines in which the mu enhancer is active.

Circular DNA is a product of the immunoglobulin class switch rearrangement

The class of immunoglobulin is defined by the constant region of its heavy chain. When a B lymphocyte switches the class of heavy chain it produces, the constant region of mu-type heavy chain is replaced; this occurs through a DNA rearrangement that brings the gene segment encoding the new constant region close to the VDJ segment encoding the variable region. The pre-B-cell line 18-81, which switches from heavy chain mu to gamma 2b production in culture, occasionally abnormally rearranges the heavy chain locus so that DNA sequences between the switch regions of mu and gamma 2b are inverted. Because looping-out is an intermediate step in generating an inversion, the switch rearrangement could occur by looping-out and deletion. Provided that recombination is reciprocal, this would produce a circle of DNA. Indeed, circular DNA molecules have been isolated as products of rearrangement among gene segments encoding the variable regions of the T-cell receptor and of the immunoglobulin heavy chain and light chain. But whereas the breakpoints for the variable region rearrangement are precisely defined, the breakpoints for any given heavy chain class switch are scattered over a length of greater than 6 kilobases, including both switch regions. We have now isolated circular DNA containing the sequences deleted by class-switching, thereby showing that the immunoglobulin heavy chain class switch occurs through looping-out and deletion.

Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line.

A murine cell line (IxN/2b) absolutely dependent upon exogenous IL-7 for continued growth has been obtained that expresses lymphoid precursor and class I MHC antigens and also contains a rearranged mu heavy chain. This cell line has been used to define the binding and structural characteristics of the murine IL-7 receptor using 125I-labeled recombinant murine IL-7. 125I-IL-7 binding to IxN/2b cell was rapid and saturable at both 4 degrees and 37 degrees C. Equilibrium binding studies produced curvilinear Scatchard plots at both temperatures with high and low affinity Ka values of approximately 1 x 10(10) M-1 and 4 x 10(8) M-1, respectively, and a total of 2,000-2,500 IL-7 binding sites expressed per cell. Experiments measuring inhibition of binding of 125I-IL-7 by unlabeled IL-7 also produced data consistent with the existence of two classes of IL-7 receptors. Evidence concerning the possible molecular nature of two classes of IL-7 receptors was provided by dissociation kinetics and affinity crosslinking experiments. The dissociation rate of 125I-IL-7 was markedly increased when measured in the presence of unlabeled IL-7 at both 37 degrees and 4 degrees C, which is diagnostic of a receptor population displaying negative cooperativity. Crosslinking studies showed that under both reducing and nonreducing conditions, the major crosslinked species observed corresponded to a receptor size of 75-79 kD while a less intense higher molecular mass crosslinked species was also seen which corresponded to a receptor size approximately twice as large (159-162 kD). Both types of experiments suggest that the IL-7 receptor may form noncovalently associated dimers in the membrane. The IL-7 receptor was expressed on pre-B cells, but not detected on several murine B cell lines or primary mature B cells. It was also expressed on murine thymocytes, some T lineage cell lines, and on bone marrow-derived macrophage. All cells binding 125I-IL-7 exhibited curvilinear Scatchard plots. No cytokines or growth factors tested were able to inhibit binding of 125I-IL-7 to its receptor. These results define the initial binding and structural characteristics, and the cellular distribution, of the murine IL-7 receptor.

In rat B lymphocyte genesis sixty percent is lost from the bone marrow at the transition of nondividing pre‐B cell to sIgM+ B lymphocyte, the stage of Ig light chain gene expression

The cycling B precursor cells in rat bone marrow (BM) that carry the B220 antigen and no surface Ig daily produce 780 million new cells. The pool of recirculating B lymphocytes in the rat, however, renew at a rate of only about 40 million cells/day. To analyze at which stages in B lymphocyte genesis the cell loss occurs, we identified post-mitotic cells in the rat BM B lineage, and determined their renewal rates. We used 5-bromo-deoxyuridine (BrdUrd) to label DNA-synthesizing cells, identifying incorporated BrdUrd with the mouse monoclonal antibody BU-1. B lineage cell subsets were identified by the markers HIS24 antigen (rat B220), terminal deoxynucleotidyl transferase (TdT), Ig mu heavy chain, and complete Ig. By use of double and triple immunocytology, we determined the extent of BrdUrd incorporation in the various B lineage compartments [HIS24+TdT-Ig-, TdT+, cytoplasmic mu chain (c mu)+ surface (s) IgM- pre-B, sIgM+ B]. Both sIgM+ B lymphocytes and all B precursors with cell diameters less than 11-12 microns were virtually devoid of DNA synthesis, as indicated by S-phase indices below 2%. In contrast, S-phase indices of large B precursors ranged between 43%-66%. We established the renewal rates of nondividing BM B lineage cells by placing osmotic minipumps containing BrdUrd subcutaneously in the flank of rats. The nondividing BM B lineage cells all renewed rapidly at rates between 2.4% and 5.6%/h, representing average half-lives of 29 to 12 h. In absolute numbers, the renewal/day/whole body BM was 165 X 10(6) for sIgM+ B lymphocytes, 422 X 10(6) for small c mu+ sIgM- pre-B cells, 89 X 10(6) for small TdT+ cells and 35 X 10(6) for small HIS24+TdT-Ig- cells. Assuming that recirculating B lymphocytes in the periphery are the descendants of BM sIgM+ B lymphocytes, which in their turn are the progeny of small pre-B cells, the renewal data indicate the following. Of the 165 million potentially available BM B lymphocytes, only 40 million cells become incorporated in the pool of recirculating B lymphocytes, representing a loss of 75%. BM B lymphocytes, in turn, use only (165/422 X 100% = ) 40% of the potential output from their immediate precursors. The 60% loss that occurs here may reflect the extent of aberrant Ig light chain gene rearrangement in normal B lymphocyte genesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Elimination of CD8+ thymocytes in transgenic mice expressing an anti-Lyt2.2 immunoglobulin heavy chain gene.

Individual T cell populations are characterized by specific surface proteins, namely by the T cell receptor complex (TCR) and by two accessory molecules, CD8 (Lyt2) and CD4 (L3T4). CD8 and CD4 are required for T cell interactions with class I or class II major histocompatibility complex molecules. In the thymus, immature CD8(-4)-TCR- cells differentiate, possibly via a short stage of CD8+4- thymocytes, into CD8+4+ TCR+ T cells and mature further into the main T cell populations, the CD8+4- TCR+ cytotoxic T lymphocytes and the CD4+8- TCR+ T helper cells. In order to analyse the differentiation steps involving CD8, we generated transgenic mice expressing mu heavy chain genes from an anti-Lyt2.2 hybridoma. Transgenic lines expressing either the complete (mu sm) or only the secreted mu protein (mu s) suffer from a severe depletion of their CD8+4+ thymocytes affecting also the mature CD8+4- and CD4+8- populations. The depletion is correlated to the expression of transgenic mu-chain proteins within thymocytes. This intrathymocyte expression of the mu chain prevents CD8-4- thymocytes from further differentiation, most probably via intracellular interactions between mu heavy chain and CD8 proteins. These results show that CD8 plays an important role during thymocyte maturation.

High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions.

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.

Dynamics of early B lymphocyte precursor cells in mouse bone marrow: proliferation of cells containing terminal deoxynucleotidyl transferase.

Three populations of early B lymphocyte precursor cells lacking mu heavy chains have been defined in mouse bone marrow, based on immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein, as detected by monoclonal antibody 14.8 (TdT+14.8- cells; TdT+14.8+ cells; TdT-14.8+ cells). We have now analyzed the frequency, size distribution, proliferation and production rates of TdT+ cells in mouse bone marrow. These formed well-defined populations of medium-sized cells, the TdT+14.8+ cells tending to be larger than TdT+14.8- cells (modal cell diameters in cytocentrifuge preparations; 10.0 microns and 9.0 microns, respectively). Some TdT+ cells (1%-2%) were normally in metaphase, the TdT being dispersed through the cytoplasm. After inducing mitotic arrest with vincristine, the incidence of TdT+ cells in metaphase increased linearly from 2 to 4 h, indicating a turnover of 5.1%/h for TdT+14.8- cells and 9.0%/h for TdT+14.8+ cells. Subtraction of turnover data for TdT+14.8+ cells from those previously obtained for 14.8+ mu- cells gave values for the population of TdT-14.8+ cells. The calculated daily turnover of cells in the three compartments increased progressively (TdT+14.8-, 2.5 x 10(6) cells; TdT+14.8+, 5.0 x 10(6) cells; TdT-14.8+, 36.0 x 10(6) cells), accompanied by a shortening of the average apparent cell cycle time (TdT+14.8-, 20 h; TdT+14.8+, 11 h; TdT-14.8+, 8 h). The results demonstrate a progressive expansion of cell production at three putatively successive stages of early B lymphocyte development before the expression of mu chains. The findings contribute to a kinetic model of primary B cell genesis in mouse bone marrow.

Interleukin 6 induces secretion of IgG1 by coordinated transcriptional activation and differential mRNA accumulation.

The molecular mechanism by which interleukin 6 (IL-6) induces terminal differentiation of B cells was investigated in a subpopulation of the clonal human B-lymphoblastoid cell line CESS selected for high density of cell surface IgG1. Induction of CESS cells with IL-6 resulted in a 15-fold preferential accumulation of secreted-specific gamma 1 (gamma 1s) mRNA but not of the alternatively processed membrane-specific gamma 1 (gamma 1m) mRNA. Similarly, microseconds mRNA but not the microns mRNA of the nonproductively rearranged mu heavy-chain allele was also increased. Accompanying the differential accumulation of gamma 1s mRNA was a 4.5-fold increase in lambda light-chain mRNA, leading to secretion of IgG1. Analyses of transcription in isolated nuclei demonstrated that transcriptional activation was the primary mechanism for quantitative increase of immunoglobulin mRNAs (5.5-fold for gamma 1 and mu and at least 2-fold for lambda). Since polymerase loading is diminished by 75% before reaching the downstream gamma 1m polyadenylylation site in CESS cells, irrespective of IL-6 induction, transcriptional pausing/termination appears intrinsic and contributes to the selection of gamma 1s and gamma 1m polyadenylylation sites in activated B cells. Furthermore, differential mRNA stabilization is likely to contribute to the alteration of the gamma 1s/gamma 1m mRNA ratio at IL-6 induction.

Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.

The conventional model of polymeric IgM depicts a unique structure in which the mu heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the mu chain. To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine. In each case the single mutants were able to assemble polymeric IgM, which was analyzed for its size, morphology, J chain content and activity in complement dependent cytolysis. Whereas normal polymeric IgM is composed predominantly of pentameric and hexameric molecules, the mutant IgM-Ser414 is covalently assembled as pentamers and smaller forms; IgM-Ser575 is assembled as covalent hexamers. IgM-Ser337 appears to include the same pentameric and hexameric forms as normal IgM except that, unlike normal polymeric IgM, most pentameric/hexameric IgM-Ser337 is not covalently assembled. J chain is present in polymeric IgM-Ser337 but absent in polymeric IgM-Ser414 and IgM-Ser575. IgM-Ser414 is defective in activating the classical pathway of complement dependent cytolysis. Our observations are consistent with models in which the covalent linkages between mu chains are mediated by disulfide bonded Cys337-Cys337, Cys414-Cys414 and Cys575-Cys575 but indicate that the arrangement of these Cys-Cys pairs in series and in parallel varies among and within IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymemse Chain Reaction Using Mixed Primers: Cloning of Human Monoclonal Antibody Variable Region Genes from Single Hybridoma Cells

We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies. (Less)

A repertoire of monoclonal antibodies with human heavy chains from transgenic mice.

The introduction of human immunoglobulin gene segments in their unrearranged configuration into the germ line of mice might allow the production of a repertoire of human antibodies. Such transgenic mice could be used for the production of human monoclonal antibodies against human antigens. To test the feasibility of this approach, mice were created that carry a human heavy-chain minilocus comprising unrearranged immunoglobulin variable, diversity, and joining elements linked to a human mu-chain gene. The gene segments of this minilocus are rearranged in a large proportion of cells in thymus and spleen but not in nonlymphoid tissue. Some 4% of the B lymphocytes synthesize human mu chains resulting in a serum titer of about 50 micrograms of transgenic IgM antibody per ml. Hybridomas were established from the transgenic mice that stably secreted several micrograms of antibodies containing human mu heavy chains per milliliter.

Highly efficient neutralization of HIV with recombinant CD4-immunoglobulin molecules

The human immunodeficiency virus type 1 (HIV-1) exploits the cell surface CD4 molecule to initiate the infection which can lead, eventually, to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope protein, gp120, interacts specifically with CD4 and soluble CD4 molecules have been shown to inhibit HIV infectivity in vitro. Effective inhibition in vivo may, however, require more potent reagents. We describe here the generation of molecules which combine the specificity of CD4 and the effector functions of different immunoglobulin subclasses. Replacing the VH and CH1 domains of either mouse gamma 2a or mu heavy chains with the first two N-terminal domains of CD4 results in molecules that are secreted in the absence of any immunoglobulin light chains. We find that the pentameric CD4-IgM chimaera is at least 1,000-fold more active than its dimeric CD4-IgG counterpart in syncytium inhibition assays and that effector functions, such as the binding of Fc receptors and the first component of the complement cascade (Clq), are retained. Similar chimaeric molecules, combining CD4 with human IgG were recently described by Capon et al., but these included the CH1 domain and did not bind Clq. Deletion of the CH1 domain may allow the association and secretion of heavy chains in the absence of light chains, and we suggest that the basic design of our constructs may be generally and usefully applied.