Biomedical subjects

R B Bankert

Publications and source records attributed to R B Bankert.

At least 37 records · Page 2Linked to original sources

PubMed1996

VH and VL gene complexes encoding an anti-spectrin antibody are defined by nucleotide sequencing of cDNA from a hybridoma generated from Hu-PBL-SCID mouse.

Previously we reported that human imunocompetent cells engrafted into scid mice mount a sustained and vigorous humoral immune response to murine erythrocytes. One of the dominant and consistently observed reactivity pattern of these antibodies in immunoblot analysis is with the alpha and beta isoforms of spectrin. In order to define the human xenoreactive response more completely, a hybridoma was generated (from a hu-PBL-scid mouse) whose antibody reacted with two high molecular weight species 225 to 250 kDa. We report here that this conserved antibody species reacts with both the murine and human erythrocyte proteins and cDNA nucleotide sequence analysis of the light and heavy chain genes encoding this antibody reveals that the light chain variable region gene has been previously observed in association with an autoreactive antibody. In addition to characterizing a conserved human B cell clonotype this is the first report of a human monoclonal antibody being generated from the hu-PBL-scid model using the standard hybridoma technology.

Amino Acid Sequence↗

PubMed1996

In vivo cytokine gene therapy of human tumor xenografts in SCID mice by liposome-mediated DNA delivery.

The human interleukin-2 (IL-2) gene was successfully delivered into established human tumor xenografts in SCID (severe combined immunodeficient) mice by cationic liposome-mediated DNA delivery. A bicistronic mammalian expression vector containing a reporter gene (beta-galactosidase) and human IL-2 cDNA was complexed with either lipofectin or DC-cholesterol liposomes and transferred to tumor xenografts by direct intratumoral injection. Transfection of tumors was confirmed by staining of tumor sections for beta-galactosidase activity and by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of IL-2 mRNA. Growth suppression of tumor xenografts was observed in animals injected with plasmid-liposome complexes but not in animals that received liposomes or naked plasmid only. Complete tumor regression, mediated by the mouse natural killer cells, was observed in 50-80% of the mice treated with the plasmid containing the IL-2 cDNA. The effectiveness of the treatment was dependent on the transfection efficiency and the tumor size at the start of therapy. An initial IL-2 independent suppression of tumor growth was also observed with a plasmid carrying only the beta-galactosidase gene but this effect was temporary and did not lead to tumor regression. These results establish that human tumor xenografts growing in SCID mice can be transfected in vivo by liposome mediated gene delivery and that both IL-2-dependent and IL-2-independent factors may contribute to the tumor suppression observed here.

Animals↗

PubMed1995

Human antibody response in human peripheral blood leukocyte/severe combined immunodeficient chimeric model is dependent on B and T cell costimulation via CD40/CD40 ligand.

We have used a human/severe combined immunodeficient (SCID) mouse chimeric model (in which human PBLs are engrafted into SCID mice) to investigate in vivo the role of the CD40/CD40 ligand (CD40L) interaction in the generation of humoral immunity by engrafted human cells. It is established in this work that the thymic dependent, multiclonal, humoral immune response of human lymphocytes to mouse erythrocyte protein Ags is inhibited significantly when CD40/CD40L ligation is prevented in vivo. Suppression of the response was observed with administration of neutralizing Abs specific for either CD40 or CD40L, or with the soluble fusion protein human CD40-Fc. Human anti-mouse erythrocyte Abs and the total human Ig levels in the sera of the SCID mice were suppressed for up to 10 wk when the neutralizing agents were administered at the time of human leukocyte engraftment, i.e., coincident with Ag stimulation, and subsequently at 2 and 4 days after Ag stimulation. These results are the first to demonstrate the ability of CD40/CD40L blocking agents to inhibit the human Ag-specific humoral immune response in vivo, and they sustain the notion that these blocking agents may be utilized clinically to eliminate immune responses associated with autoimmunity or allergy. In addition to confirming the importance of the CD40/CD40L interaction of T and B cells in vivo, it is established in this work that the mouse erythrocyte-specific humoral immune response of the lymphocytes in the human PBL/SCID mouse model is a bona fide T cell-dependent response, and that this is a viable model with which to study human lymphocyte activation in vivo. These results also suggest that the costimulatory activation of human B and T cells is not dependent on the microenvironment of the germinal centers.

Animals↗

PubMed1995

Allelic differences in the VHOx-1 gene explain the absence of a B cell clonal dominance in the primary response of C57BL/6 mice to phthalate.

A highly conserved Id (CRIXmp-1) associated with the murine (BALB/c) humoral immune response to the hapten phthalate (Xmp) is conspicuously absent in C57BL/6 mice. The absence of this Id in C57BL/6 mice is shown here to be due to the absence of the appropriate VH gene (VHOx-1) usage in the Xmp response. To determine whether the failure to utilize this VH was due to an active suppression or to the lack of the requisite VH gene in the available repertoire, VHOx-1 gene-specific primers were used to amplify the germ-line VHOx-1 gene from genomic DNA from BALB/c and C57BL/6 mice. The germ-line coding sequence of the C57BL/6 allele of the VHOx-1 gene is 99% similar to the germ-line coding sequence of the BALB/c allele. Amplification of cDNA made from splenic RNA from C57BL/6 mice confirmed that this gene is expressed. There are four nucleotide differences that lead to three amino acid changes in the predicted protein sequence. Each change is either in or immediately adjacent to a complementarity-determining region (CDR). Two of these changes are unique to the C57BL/6 allele and are not shared with CRIXmp-1-expressing strains. These two changes are predicted to alter the Xmp binding capabilities of the C57BL/6 allelic form of this VH gene, thereby explaining the absence of the Xmp-1 clonotype, which is dominant in the primary Xmp immune response of most other strains of mice.

Alleles↗

PubMed1995

Transient dominance of the early primary immune response by a highly conserved B-cell clone that is distinguished by its lack of memory, high threshold of activation, and a high affinity.

We establish here that the very early primary response to the hapten phthalate (Xmp) is distinguished by a restricted heterogeneity with over 80% of the anti-Xmp antibodies expressing a single well-defined cross-reactive idiotype (CRIXmp-1) associated with a previously described highly conserved clonotype that is expressed by most inbred strains of mice and many outbred mouse populations as well. The characteristic early dominance of this one clonotype in the primary response is transient. While the CRIXmp-1 clonotype is present later in the primary and throughout the secondary response, it represents only a very small proportion of the total anti-Xmp antibody population at these times. The early dominance of the single clonotype is rapidly replaced by a heterogeneous population of antibodies. Differential activation thresholds for the primary response clonotype (CRIXmp-1) and secondary response clonotypes, and the failure of the dominant primary response clonotype to expand in the secondary response (i.e., absence of memory) suggest the presence of two distinct B-cell lineages.

Animals↗

PubMed1994

Generation of monoclonal antibodies that distinguish between mesotheliomas and other tumor of the lung.

The accurate diagnosis of mesothelioma remains difficult despite advances of diagnostic technique. And specific monoclonal antibody (McAb) against mesothelioma have not been reported. In an attempt to develop mesothelioma specific McAb(s), spleen cells from a mouse immunized with isolated tumor cells were fused to a drug resistant mouse myeloma cell lines. Over 200 hybridomas were assayed for their preferential reactivity with mesothelioma cell lines or mesothelioma tumor biopsy tissues. Two monoclonal antibodies 2A3 and 4E1 were identified that bound 6/7 of the mesotheliomas, tested, but did not bind to the majority, 11/13 (for 2A3) and 12/13 (for 4E1), of other lung tumor types. Based upon western blot analysis of one and two-dimensional gels and upon the distribution pattern of the antibody recognized molecule in mesotheliomas and non-mesothelioma lung tumors, 2A3 binds to the cell adhesion molecule CD44. While the specificity of 4E1 has not yet been unequivocally established it appears to recognize a variant form of the CD44 molecule.

Animals↗

PubMed1994

Human villous adenomas engrafted into scid mice survive for prolonged period without malignant transformation.

Human villous adenomas are thought to represent premalignancies that subsequently give rise to colorectal adenocarcinomas. Currently there is no in vivo model in which to study the dedifferentiation and malignant transformation of these tumors. We establish here that human villous adenomas can be successfully engrafted into severe combined immunodeficient (scid) mice. Furthermore, these xenografts remain viable for up to 18 mo after either a subcutaneous or intraperitoneal inoculation of the human tissue. Tumors grew slowly and secreted a clear mucinous fluid. Examination of the tumors histologically at 1, 4, and 12 mo after implantation revealed that the villous polypoid structure was maintained and islands of atypical cells were observed within pockets of mucin surrounding the adenomatous tissue. No gross or histologic evidence of malignancy was detected throughout the 20-mo observation period. The human identity of the cells in the graft was confirmed by DNA in situ hybridization with a human-specific probe. We conclude that the human-scid xenograft described here represents a viable animal model with which to study the potential malignant dedifferentiation of villous adenomas over a prolonged period of time and to evaluate the possible contribution of selected oncogenic vectors on the malignant transformation of these adenomas.

Adenoma, Villous↗

PubMed1994

Clones of tumor cells derived from a single primary human lung tumor reveal different patterns of beta 1 integrin expression.

Previously we reported that over 75% of human non-small cell lung cancers overexpress the beta 1 integrin VLA-2 on their surface and show an increase in the mRNA encoding the alpha-2 chain of this integrin. These results suggested the possibility that the overproduction and overexpression of one or more of the beta 1 integrin may be involved in the pathogenesis of human lung tumors by modulating the invasive and/or metastatic potential of the tumor. We report here the generation and characterization of multiple clones of tumor cells derived from the primary culture of cells obtained from biopsy tissue of an aggressive human squamous cell lung tumor. We show that these tumor clones (or clonotypes) exhibit seven different yet stable phenotypes with respect to the expression of five members of the beta 1 integrin family. These results illustrate that a primary human lung tumor consists of multiple subpopulations of cells that while indistinguishable by ultrastructure are heterogeneous with respect to their beta 1 integrins. The availability of these distinct tumor clonotypes derived from a single tumor biopsy have made it possible to test the assumption that the beta 1 integrins play a role in tumor progression. The feasibility of this approach is demonstrated here by the intravenous inoculation of different human tumor clonotypes into severe combined immunodeficient (scid) mice. Our preliminary results with a pair of tumor clonotypes differing in VLA-1 and VLA-2 expression level reveal that the clonotype with high level of VLA-1 and VLA-2 displays a substantial increase in the experimental engraftment and metastasis of the human tumor cells in scid mice.

Animals↗

PubMed1993

Arrest of human lung tumor xenograft growth in severe combined immunodeficient mice using doxorubicin encapsulated in sterically stabilized liposomes.

Incorporation of polyethylene glycol-derivatized phospholipids into liposomes results in carriers that can enhance the therapeutic efficacy of encapsulated drugs by imparting the ability to evade the reticuloendothelial system and remain in the circulation for prolonged periods. In this study, doxorubicin encapsulated in these sterically stabilized liposomes (S-DOX) is shown to completely arrest the growth of human lung tumor xenografts in severe combined immunodeficient (scid) mice. Doxorubicin administered at equivalent doses as free drug or encapsulated into conventional liposomes was ineffective at completely arresting the growth of this human tumor, although a decrease in tumor growth rate compared to untreated controls was observed. Scid mice were found to be significantly more susceptible to the toxic effects of doxorubicin than were immunocompetent C.B-17 control mice, a characteristic that is likely to result from the deficit in DNA repair mechanisms previously identified in scid mice. However, doxorubicin toxicity in scid mice could be minimized while maintaining the antitumor activity of doxorubicin encapsulated in sterically stabilized liposomes by administering the drug in multiple weekly injections at low doses. This report provides the first evidence that antitumor drugs delivered in sterically stabilized liposomes are more effective at arresting the growth of human tumors than are conventional delivery systems. In addition, the scid mouse is presented as a viable model in which to study novel chemotherapeutic approaches to the treatment of human cancer.

Animals↗

PubMed1993

The study of human neoplastic disease in severe combined immunodeficient mice.

Experimental models of human neoplastic diseases are used in attempts to reconstruct events that occur in patients with cancer. Although in vitro systems provide a wealth of information about the cellular and molecular biology of tumor cells, they are inadequate for studies that address the complexities of human neoplasia pertaining to metastasis, experimental therapeutics, and immunity. Since the late 1960s, athymic nude mice have provided an opportunity to study the growth and, in some cases, the metastasis of xenografted human tumors in vivo. Recently, the availability of severe combined immunodeficient (scid) mice has provided an alternative model for studying human malignancies and has led to numerous reports of its distinct advantages over nude mice. When direct comparisons have been made, it has often been apparent that human tumors grow better and are more likely to metastasize in scid mice than in nude mice. Indeed, some human tumors which have never before been propagated in vivo will engraft in scid mice. Furthermore, the unique capability of scid mice to support human immunocompetent cells offers the potential to study the human immune response to tumors. This article outlines the current progress toward defining and using scid mice in models of human neoplastic disease. Characteristics which distinguish scid mice from nude mice are emphasized and a discussion of future prospects is included.

Animals↗

PubMed1992

Nucleotide sequence of messenger RNA encoding VHDJH and VKJK of a highly conserved idiotype-defined primary response anti-hapten antibody.

The primary humoral immune response of mice to the hapten phthalate (Xmp) is focused upon two adjacent immunodominant negatively charged carboxyl groups on a benzene ring that are in positions meta and para to the azolinkage (i.e., Xmp) to the protein carrier keyhole limpet hemocyanin. A significant fraction of the anti-Xmp antibodies raised in several different inbred mouse strains (BALB/c, DBA/2, A/HeHa; C3H, and SM/J), and many wild mouse populations express a cross-reactive Id, CRIXmp-1. This CRIXmp-1 is conspicuously absent in C57BL/6 mice. In order to obtain a better understanding of the events and parameters that influence the selection and regulation of the primary response B cell repertoire, and to explore the structural basis of Ag binding, we have determined the nucleotide sequence of the entire V region gene complexes, which encode the H and L chains of these highly conserved and dominant CRIXmp-1+ antibodies. Our data establish that the H chain gene complex consists of a single VH germ-line gene that is identical to VH Oxazolone-1, encoding the H chain of another highly conserved and dominant cross-reactive Id family associated with the primary response to Oxazolone. In CRIXmp-1+ Xmp-specific hybridomas this gene is joined to a limited set of D region sequences that express a conserved amino acid motif-GLR. At least three of the five D regions examined are coded for by DFL16.2. This VHD complex can be utilized with one of three different JH region genes (JH1, JH2, and JH4) without any significant effect upon antibody fine specificity or Id. In spite of this lack of JH fidelity all of the CRIXmp-1+ hybridomas have precisely maintained the same length in the H chain CDR3 and FRW4 by altering either the length of the D segment or the length of JH. Nucleotide sequence analysis of the VL gene complex of CRIXmp-1+ anti-Xmp antibodies indicates that the L chain V region is also encoded by a single germ-line gene. The amino acid sequence predicted from the nucleotide sequence of the VKJK from Xmp-specific CRIXmp-1+ hybridomas is identical to the sequence of the anti-arsonate antibody 1210.7, which is the prototype of another Id family (CRI) that is conserved and dominant in BALB/c mice.

Acute-Phase Reaction↗

PubMed1992

Engraftment of human peripheral blood leukocytes into severe combined immunodeficient mice results in the long term and dynamic production of human xenoreactive antibodies.

Severe combined immunodeficient (SCID) mice engrafted with human peripheral blood leukocytes (hu-PBL-SCID) represent a potentially important small animal model for the study of human immune function. Attempts to generate human primary immune responses to exogenous Ag in the hu-PBL-SCID have had limited success which raises questions about the functional capacity of human lymphocytes in the SCID environment. Here, we demonstrate that the spontaneously secreted human Ig in hu-PBL-SCID includes antibodies with specificity for several different mouse RBC (mRBC) proteins. These antibodies apparently reflect the transfer of peripheral B cells which are responsible for the production of naturally occurring xenoreactive antibodies in the donor. Western blot analysis showed that engraftment of anti-mRBC specificities was random among mice receiving PBL from the same donor sample. In at least one mouse, this engraftment was polyclonal and included human IgM and IgG which recognized at least 12 different mRBC proteins ranging in size from 35 to > 200 kDa. Anti-mRBC specificities were found to vary with time demonstrating a dynamic expression of the human xenoreactive repertoire in hu-PBL-SCID. In contrast to mice engrafted with human PBL, mice engrafted with another source of human B cells, i.e., tumor-infiltrating leukocytes, produced very little or no human anti-mRBC antibody. Ag-driven proliferation of xenoreactive clones may result in a skewing of the engrafted human B cells in hu-PBL-SCID which could account in part for the limited ability of hu-PBL-SCID to respond to exogenous Ag. The long term production of anti-mRBC antibodies and the modulation of the expressed xenoreactive repertoire observed in hu-PBL-SCID represents an opportunity to study the molecular genetics and cell biology of the human humoral immune response to a defined complex Ag.

Adsorption↗

PubMed1992

Translocation of spectrin and protein kinase C to a cytoplasmic aggregate upon lymphocyte activation.

We have previously reported that mammalian tissue lymphocytes exhibit significant heterogeneity with respect to the subcellular distribution of spectrin and that this phenomenon may result from a dynamic behavior of spectrin in response to activation signals. Here, we further characterize the involvement of spectrin in lymphocyte activation by examining its relationship with protein kinase C (PKC). PKC isoenzymes are a family of cytosolic kinases that translocate from the soluble to particulate fraction upon cell stimulation. It is reported here that activation of lymph node T cells through the antigen-specific receptor, or direct activation of PKC by phorbol esters, results in a striking increase in cells expressing a cytoplasmic aggregate of spectrin. Additionally, a concurrent increase in cells expressing aggregates of the beta II isozyme of PKC is observed. Immunofluorescence staining revealed that spectrin and PKC beta II are colocalized in untreated lymphocytes and that these two proteins are coincidently translocated to the same focal aggregate within the cytoplasm following stimulation. This redistribution of spectrin and PKC beta is blocked by pretreatment with calphostin C, a specific inhibitor of PKC. Solubility studies showed that there is an increase of both proteins in the detergent-insoluble fraction of lymphocytes upon activation, and immunoprecipitation studies indicated that the soluble form of these molecules may be associated directly or indirectly as part of a complex of proteins. These data indicate that the positioning of the spectrin-based cytoskeleton is sensitive to activation signals and may play a role in the function or positioning of PKC beta II.

Animals↗

PubMed1991

COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells.

Transfectants of mature B cell lines that bind phosphorylcholine were made in order to understand the role of the COOH terminus of the mu chain of membrane IgM (mIgM) in generation of antigen-specific signals. A chimeric receptor (I-A alpha tail) was constructed by replacing 40 amino acids from the mu COOH terminus with that of major histocompatibility complex class II I-A alpha chain. The effect of wild-type and chimeric tails were studied on representative immediate-early antigen-specific signals. The I-A alpha tail hybrid, but not the wild-type receptor, was defective in antigen-driven Ca2+ mobilization, although it could effectively endocytose ligand-receptor complexes. Signal(s) transduced through the wild-type receptor led to transient induction of selected immediate-early gene messages (Egr-1, c-fos, Jun) above basal levels. However, the signal(s) generated after crosslinking of the I-A alpha tail receptor either showed no effect (c-fos) or actually repressed basal level expression of Egr-1 and Jun. Thus, we have established that receptor-mediated endocytosis can be distinguished from other early events associated with B cell activation, based on their differential dependence upon the structural fidelity of the COOH-terminal sequence of mIgM.

Amino Acid Sequence↗

PubMed1991

Human lung tumor-associated antigen identified as an extracellular matrix adhesion molecule.

A single chain glycoprotein with an estimated molecular mass of 160 kD (gp160) was previously identified as a human lung tumor-associated antigen. This tumor marker is shown here to be associated noncovalently with a second 130-kD protein. Sequential immunoprecipitation studies of surface iodinated lung tumor cell lysates reveal that this heterodimeric complex is indistinguishable serologically and structurally from the integrin VLA-2, found originally on activated T lymphocytes and platelets. The VLA-2-like complex expressed on the lung tumors possesses similar characteristic Mg2+ dependent binding of collagen and laminin as observed with VLA-2 on normal cells. RNA analysis indicates that human lung tumors express at least 20 times more VLA-2 alpha chain message than normal adult human lung tissue. The results presented here raise the possibility that the overproduction of VLA-2 may be involved in the pathogenesis of human lung tumors by modulating the invasive and metastatic potential of the tumor.

Antigens, Tumor-Associated, Carbohydrate↗

PubMed1991

Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.

The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.

Adult↗

PubMed1990

Vaccination with membrane-associated idiotype provides greater and more prolonged protection of animals from tumor challenge than the soluble form of idiotype.

The purpose of this work was to compare the efficacy of immunizing mice with a soluble vs a cell-associated form of a tumor Ag. A murine B cell tumor (2C3), which displays an Id on its cell surface, grows progressively and gives rise to Id-negative tumor variants in nonimmunized animals. We previously reported that the tumor variants arise as a consequence of Id-specific T cell suppression of the Id+ tumor. The Id-specific effector T cells are CD4+, CD8-. Based upon these findings vaccination protocols have been designed and tested to determine whether the expansion of tumor-specific effector T cells would eliminate the Id+ tumors and prevent the subsequent generation of Id- tumor variants in vivo. MHC-restricted T cells typically recognize soluble Ag subsequent to modification by an APC, and APC may ultimately express a processed form of the Ag that is different from that expressed on the surface of the tumor cells. Based upon this assumption, the efficacy of immunizing mice with cell-associated 2C3 Id was compared to immunization with a soluble form of the same Id. Mice were immunized with either irradiated 2C3 cells or syngeneic spleen cells to which 2C3 protein was covalently linked. These immunization protocols provided a complete and lasting protection against a tumor challenge of up to 1 x 10(6) tumor cells. In contrast, most mice hyperimmunized with a soluble form of the Id did not survive this level of tumor challenge in spite of the production of significant levels of anti-Id antibodies. Mice immunized with the soluble form of the Id, which did survive, produced slowly progressing tumors expressing a 1000-fold less of the marker Id. These results illustrate the importance of understanding and properly exploiting a host's natural response to a tumor-specific Ag when designing effective immunization protocols for cancer therapy.

Animals↗

PubMed1989

Endocytosis of antigen, anti-idiotype, and anti-immunoglobulin antibodies and receptor re-expression by murine B cells.

The endocytosis of Ag mediated by membrane-associated Ig (mIg) molecules has been spectrophotometrically monitored using a cell line (2C3) specific for the hapten phthalate (Xmp) and employing conjugates of Xmp and horseradish peroxidase (HRP) as the labeled ligand. Approximately 50% of both Xmp-HRP, or the larger ligand, Xmp-keyhole limpet hemocyanin-HRP, are internalized rapidly, reaching an initial plateau by 30 min. The rate of endocytosis of anti-idiotype-HRP is similar to the rates that were observed for the hapten-bearing ligands, while a slower rate of endocytosis of anti-Ig-HRP was observed. The percent of ligand bound that is internalized and the rate of endocytosis appear to be largely independent of the size and amount of ligand bound per cell. However, mIg-mediated endocytosis is markedly reduced when mIg-ligand complexes are more extensively cross-linked by the binding of a second antibody. In addition to the initial rapid phase of endocytosis, there is a prolonged phase during which more of the bound ligand is internalized, and up to 90% of the internalized ligand is degraded. Re-expression of Ag-binding receptors by the 2C3 cells is independent of new protein synthesis and is accomplished in part by the translocation of a presynthesized pool of mIg molecules from the cytoplasm to the plasma membrane of the cell. The kinetics of endocytosis of HRP-labeled anti-Ig antibodies by BALB/c splenic B-lymphocytes and other B-lymphocyte cell lines is very similar to the endocytosis of Ag and anti-idiotype observed with the 2C3 cell line. Light and electron microscopy are also performed to visually confirm that the HRP-labeled ligands are being internalized and to determine the percentage of cells involved in this process. Finally it was determined that the transmembrane and cytoplasmic domains of the mIg molecules are required for endocytosis since the secreted form of the molecule (which lacks these domains) fails to mediate the internalization of bound ligand.

Animals↗