Selection of antigen-specific, idiotype-positive B cells in transgenic mice expressing a rearranged M167-mu heavy chain gene.

Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.

Antigen binding and idiotype analysis of antibodies obtained after electroporation of heavy and light chain genes encoding phosphocholine-specific antibodies: a model for T15-idiotype dominance.

Antibodies bearing the T15 idiotype dominate the murine primary immune response to phosphocholine (PC). Analysis of antigen binding of antibodies derived from V1:DFL16.1:JH1 (VH1) germline and N region-derived variant heavy (H) chains and kappa 22, kappa 24, and kappa 8 light (L) chains demonstrates that the T15H:kappa 22L (T15) antibody binds PC at least 20-40 times better than other antibodies derived from alternate germline forms of the VH1 H chain and kappa 22, kappa 24, or kappa 8 L chains. To achieve affinities in the same range as the T15 antibody, kappa 24 and kappa 8 L chain-containing antibodies must have H chains derived from variant N region or somatically mutated VH1 genes. Single amino acid differences at the VD junction of the various germline and N region variant VH1 H chains dictate the L chain that can associate with the H chain to produce a PC-specific antibody. Several H:L combinations give rise to T15 or M167 idiotype-positive antibodies that lack specificity for PC, and single amino acid substitutions or insertions at the VH1:D junction result in the loss of T15 or M167 idiotopes. Based on these observations, our data support a molecular model involving both preferential gene rearrangement and antigen-driven B cell selection to explain T15 idiotype dominance in the immune response to PC. In the absence of N region diversification, large numbers of neonatal B cells bearing the T15H:kappa 22L surface immunoglobulin M (sIgM) receptors would be selected and expanded by autologous or environmental PC antigen into the long-lived peripheral B cell pool.

GRS, a novel member of the Bcl-2 gene family, is highly expressed in multiple cancer cell lines and in normal leukocytes.

Our laboratory previously described the independent isolation of the fibroblast growth factor 4 (FGF-4) gene by NIH3T3 transformation assay using DNA from a patient with CML leukemia (Lucas et al., 1994). The FGF-4 gene was truncated by DNA rearrangement with a novel gene named GRS. In this manuscript we describe isolation of GRS cDNA and show by sequence comparison that GRS is a novel member of the Bcl-2 gene family. Northern analysis shows expression of the gene in normal human tissue to be largely restricted to the hematopoietic compartment. Analysis of the pattern of gene expression in cancer cell lines demonstrates GRS is expressed in hematopoietic malignancies and in melanoma. The chromosomal location of GRS has also been determined. The gene is positioned on chromosome 15 within bands q24-25.

Recombinant human IL-7 administration in mice affects colony-forming units-spleen and lymphoid precursor cell localization and accelerates engraftment of bone marrow transplants.

Murine reconstitution assays were used to investigate the effects of recombinant human interleukin-7 (rhIL-7) on myeloid and lymphoid precursors and on bone marrow engraftment. Reconstitution with bone marrow from rhIL-7-treated mice results in a 3.4-fold decrease in total colony-forming unit-spleen (CFU-S) activity (day 9) and an 18.1- and 11.9-fold decrease in its ability to generate thymocytes and splenic B lineage cells, respectively. In contrast, after reconstitution with splenocytes from rhIL-7-treated mice, CFU-S activity increased 23.6-fold (day 9) and the thymocyte and splenic B lineage cell regenerative capacity increased by 4.0- and 3.2-fold, respectively. In addition, CD43low+, B220low+ cells that contain pre-pro-B cells and pro-B cells were expanded two- to threefold and Ig mu-, B220+, CD2- and Ig mu-, B220+, CD2+ B lineage cells were expanded approximately 10-fold and 10- to 45-fold (depending on the tissue examined), respectively, after rhIL-7 treatment. Administration of rhIL-7 to irradiated mice transplanted with bone marrow resulted in accelerated T cell and B cell reconstitution by up to 2-4 weeks. Thus, rhIL-7 administration affects the distribution of myeloid and lymphoid precursors. Moreover, rhIL-7 administration accelerates murine bone marrow cell engraftment and therefore may be useful in reducing the engraftment time in bone marrow transplant patients.

H-2 hierarchy in the IR-gene controlled responses to hapten-modified mouse serum albumin.

Five hapten-modified autogenous mouse serum albumins (MSA), TNP11MSA, FITC8MSA, DNP64MSA, DNP5MSA, and PC2MSA, were tested for their ability to induce hapten-specific antibody responses in H-2 congenic mice of the H2d, H-2b, H-2a, and H-2f haplotypes. Four of the five modified MSAs, TNP11MSA, FITC8MSA, DNP64MSA, and PC2MSA, stimulated responses found to be under H-2-linked Ir gene control, whereas DNP5MSA failed to induce responses in any strain tested. H-2d, H-2D, and H-2a mice responded to FITC8MSA; H-2d and H-2b responded to TNP11MSA; and only H-2d mice were high responders to DNP64MSA and PC2MSA. H-2f mice failed to respond to any of the stimulating antigens. Thus, a hierarchy of H-2 haplotypes was observed in the responsiveness to modified MSA with H-2d greater than H-2b greater than H-2a greater than H-2f. Primed lymph node cells from PC2MSA immunized H-2d mice were challenged with antigen in vitro to assess the nature of the determinant recognized by T cells in the response to modified MSA. Lymph node cells proliferated only when challenged with PC2MSA. Unmodified MSA or TNP11MSA did not stimulate a proliferative response, suggesting that the immune T cells recognize a neodeterminant present on the modified PC2MSA.

Anti-idiotype monoclonal antibodies specific for the MOPC167 anti-phosphocholine transgene-encoded antibody.

Four rat x mouse hybridomas secreting monoclonal anti-idiotypic (anti-Id) antibodies (MAb) specific for the transgene-encoded antibody of the 207-4 transgenic mouse line, which carries the VH1/V kappa 24 gene segments of the IgA, phosphocholine-(PC) specific MOPC167 myeloma, were developed from a fusion of Ag8-X63.653 mouse cells with spleen cells from a rat immunized with MOPC167 and HPCM27 anti-PC antibodies. The anti-Id MAb were shown by ELISA to be specific for PC-binding proteins of VH1/V kappa 24 H and L chains of various isotypes. They did not bind VH1/V kappa 22, VH1/V kappa 8, or VH1/V kappa 1 PC-binding proteins or other IgA or IgM myeloma proteins. Analysis by flow cytometry demonstrated that these MAb bind to the transgene-encoded membrane immunoglobulin (sIgM) as expressed on > 95% of the B220 positive 207-4 spleen cells. All four MAb were able to inhibit the binding of MOPC167 to PC conjugated to bovine serum albumin. Differences in fine specificity of binding were demonstrated by differential staining of spleen cells of the 216-7 mu kappa delta Mem MOPC167 transgenic mice. In these mice endogenous H chains associate with the transgene encoded L chain to form MOPC167 crossreactive idiotopes. Two of the MAb, 28-4-3 and 28-6-20, stained significant numbers of cells, while MAb 28-5-15 did not bind to 216-7 cells. Three of the MAb, 28-5-15, 28-6-20, and 28-4-3, when conjugated to Sepharose beads, were able to induce DNA synthesis in cultures of 207-4 transgenic spleen cells. None of the MAb were able to induce an antibody response in vivo. These MAb should prove useful in staining PC-transgenic B cells for flow cytometry studies and in defining early cellular events in the activation of idiotype positive B cells by anti-Id antibodies.

Regulation of T15 idiotype dominance. III. Phosphocholine-specific B-cell activation in vivo requires major histocompatibility complex-restricted T-cell help.

We have examined the requirement for major histocompatibility complex (MHC)-restricted T-cell help in the secondary in vivo antibody response to phosphocholine (PC). The memory response to PC has been demonstrated previously to be comprised of T15-dominant IgM and IgG3 plaque-forming cells (PFC) derived primarily from the Lyb-5+ B-cell subset, and IgG1 and IgG2 PFC, few of which bear the T15 idiotype and are predominantly derived from the Lyb-5- B-cell subset. Using carrier-primed (A X B)F1 T cells which have matured in a parentA chimeric environment so that "self" recognition is of the MHC determinants of parentA but not parentB, we have found that parentA PC-primed B cells, but not parentB PC-primed B cells, are activated. Even in the presence of an ongoing parentA anti-PC response, parentB PC-primed B cells were not activated, indicating that the restriction was between the helper T cell and the B cell, not between T-helper and accessory cells. MHC-restricted T-cell help was required by B cells producing T15+ and T15- IgM, IgG3, IgG1, and IgG2 responses.

Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0.

Dominant negative or trans-dominant mutants of viral proteins represent a new and exciting potential approach to antiviral therapy. Unfortunately, the extreme specificity of a given dominant negative mutant limits its general utility in treating a broad spectrum of viral diseases, since it can typically interfere with the activity of only a single viral polypeptide encoded by a single virus. However, it seems likely that dominant negative mutants of promiscuous viral trans-activator proteins, which by definition would repress rather than activate gene expression, should be able to inhibit infectious virus production for a number of different viruses. One such dominant negative mutant, derived from the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0, was found previously to behave as a powerful repressor of gene expression from an assortment of HSV-1 and non-HSV-1 promoters in transient expression assays. In the present study, this ICP0 mutant was found to be capable of inhibiting the replication of both HSV-1 and a completely unrelated virus, human immunodeficiency virus, in cell culture. The properties of this dominant negative mutant indicate that it may have potential as a means of treating diseases caused by a number of DNA and RNA viruses. Moreover, a truncated form of ICP0 which can hypothetically be created by alternative splicing was found to possess similar inhibitory capabilities, suggesting that a virus-encoded version of this dominant negative mutant may play a role in down-regulating HSV-1 gene expression during infection in vivo.

Upstream stimulatory factor family binds to the herpes simplex virus type 1 latency-associated transcript promoter.

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is the only viral promoter that exhibits detectable transcriptional activity during a latent HSV infection. The LAT promoter-binding factor (LPBF) regulatory sequence (nucleotides -65 to -72 relative to the transcriptional start site of the 8.3-kb primary transcript) closely resembles the core recognition sequence required for binding members of the upstream stimulatory factor (USF)/major late transcription factor (MLTF) family. In this analysis, we demonstrate that oligonucleotides containing either the LPBF recognition sequence or the USF/MLTF recognition sequences from previously described promoters bind cellular factors which exhibit very similar mobilities in electrophoretic mobility shift (EMS) analyses. We also observe a high degree of similarity in competition profiles obtained in competition EMS analyses utilizing oligonucleotides containing recognition sequences for either LPBF or USF/MLTF. Furthermore, antibody supershift EMS analyses have demonstrated that the factors binding the LPBF or USF/MLTF recognition sites in these oligonucleotides are antigenically related, if not identical, and that greater than 90% of the LPBF-binding activity is antigenically related to USF. In addition, we demonstrate that both forms of in vitro translated USF proteins (43 and 44 kDa) bind to the LPBF recognition sequence within HSV-1 LP1. Taken together, these data indicate that USF is capable of binding to the HSV-1 LPBF recognition sequence and that USF is a major LPBF-binding activity in cells of neuronal and nonneuronal lineage. These data further support the hypothesis that USF may indeed play a significant role in the transcriptional activity of HSV-1 LP1.

A novel phosphocholine antigen protects both normal and X-linked immune deficient mice against Streptococcus pneumoniae. Comparison of the 6-O-phosphocholine hydroxyhexanoate-conjugate with other phosphocholine-containing vaccines.

A novel form of phosphocholine (PC), p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC) coupled to keyhole limpet hemocyanin (KLH) has been compared with unencapsulated, avirulent Streptococcus pneumoniae (R36a) and with the traditional thymus-dependent form of PC, diazophenylphosphocholine (DPPC)-conjugated KLH for its vaccine potential against virulent S. pneumoniae. Immunization with any of these three PC-containing Ags protects normal mice against a lethal challenge with 10(4) S. pneumoniae, whereas only EPC-KLH provides total protection to Xid mice. DPPC-KLH and unencapsulated S. pneumoniae confer less than 40% protection in Xid mice. Passive transfer of a PC-specific hybridoma Ab made from EPC-KLH-immunized Xid mice also provided protection against lethal challenge with S. pneumoniae. Protective anti-PC Ab were capable of binding to the surface of virulent bacteria, whereas anti-PC Ab incapable of binding to the bacterial surface failed to protect. Furthermore, serum Ab from EPC-KLH immunized and protected mice bound to S. pneumoniae, whereas secondary Abs from DPPC-KLH- or R36a-immunized mice failed to bind to the bacteria. EPC-KLH is potentially a vaccine candidate for pneumococcal prophylaxis in settings of immune compromise.

A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.

A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.

Helper T cell requirements for T15 idiotype expression on phosphorylcholine-specific antibodies.

The requirement for idiotype-specific T cells was investigated in the T15 idiotype-dominant T cell-dependent response of unprimed BALB/c and (BALB/c X C57BL/6)F1 B cells to phosphorylcholine (PC). It was first demonstrated that cloned keyhole limpet hemocyanin (KLH)-specific, major histocompatibility complex (MHC)-restricted T helper (Th) cells as well as heterogeneous KLH-primed Th populations were capable of generating PC-specific antibody responses in T-depleted unprimed B cell populations cultured in the presence of PC-KLH. The PC-binding antibody responses generated under these conditions were indistinguishable when assayed for carrier-hapten linkage requirements, immunoglobulin isotype (predominantly IgM) or PC affinity. Further, it was observed that the PC-binding antibodies which were generated in the presence of these two T cell populations expressed equivalently high levels of T15 idiotype. Assaying antibody and idiotype by either enzyme-linked immunosorbent assay or plaque-forming cell assay yielded similar results. Since monoclonal MHC-restricted, KLH-specific Th cells presumably lack any additional T cell populations, these results argue against an absolute requirement for anti-idiotypic Th cells in the generation of T15-dominant antibody responses.

Temporomandibular joint adaptations following maxillary osteotomy in adolescent monkeys.

The purpose of this study was to investigate the histologic appearance of temporomandibular joints of monkeys who had undergone total maxillary osteotomy. The specific aim of the study was to seek clues that might explain the inhibition in the mandibular growth 27 months following the surgery performed on adolescent monkeys. Eighteen female adolescent Macaca fascicularis monkeys were used. Eight served as control and ten were experimental. All experimental animals underwent 3.0 to 5.0 mm superior and 0.5 to 2.5 mm anterior repositioning of the maxilla. All animals were killed 27 months after the surgical procedure. Qualitative and quantitative histologic evaluations of the temporomandibular joints were done and results were compared with the cephalometric data. The findings showed no pathologic changes, although the histologic appearance of the temporomandibular joints of the experimental animals was more mature than that of the controls. The histologic results support cephalometric findings and the data are discussed in relationship to the role of maxilla, maxillary teeth, and occlusion in regulating mandibular growth.

Theiler's virus-induced demyelination in mice immunosuppressed with anti-IgM and in mice expressing the xid gene.

Intracerebral infection with Theiler's murine encephalomyelitis virus produces chronic immune-mediated demyelination in susceptible strains of mice. We examined the role of Ig in the pathogenesis of demyelination. In susceptible SJL/J mice (H-2s), suppression of B cell responses with IgG fraction of goat anti-mu (anti-mu IgG) from birth resulted in increased numbers and severity of demyelinating lesions in the spinal cord 35 days after infection. In contrast, treatment of resistant C57BL/10 (H-2b), C57BL/6 (H-2b), or B10.D2 (H-2d) mice with anti-mu IgG had no apparent effect since these mice did not develop demyelination or inflammation in the spinal cord following infection. Similar results were obtained with certain strains of B-cell deficient mice that exhibit the xid gene mutation. Male CBA/NJ (xid) showed increased meningeal inflammation and demyelination compared to male CBA/J mice. However, B6.CBAN, C3.CBAN, or C.CBAn mice showed no or minimal evidence of demyelination despite the presence of the xid mutation. In the SJL/J mouse, the majority of the humoral immune response to virus antigen was restricted to the IgG2b and IgM isotypes. These data indirectly support the hypothesis that immunoglobulins protect partially against development of virus-induced demyelination in susceptible but not resistant animals. In addition, the data argue strongly against the hypothesis that TMEV-induced demyelination is mediated predominantly by humoral autoimmune or humoral viral immune mechanisms.

Induction of phosphocholine-specific antibodies in X-linked immune deficient mice: in vivo protection against a Streptococcus pneumoniae challenge.

X-linked immune deficient (XID) mice are susceptible to infection with Streptococcus pneumoniae because they fail to mount an immune response to the immunodominant phosphocholine (PC) epitope on the bacterial cell wall. It is difficult to induce PC-specific antibodies in XID mice because PC-specific B cells expressing the T15-, M167- and M603 idiotype (Id), which provide protection against S. pneumoniae, are deleted in these mice via an antigen-specific, receptor-mediated process. In addition, the standard PC hapten, p-diazophenylphosphocholine (DPPC), induces high affinity phenylphosphocholine (PPC)-specific antibodies in XID mice, which are not protective against S. pneumoniae. We have used a novel PC hapten, p-nitrophenyl-6-(O-phosphocholine)hydroxyhexanoate (EPC), to induce PC-specific antibodies in XID mice. The immune response to EPC-keyhole limpet hemacyanin (KLH) is dominated by IgG1, VH1+, T15-Id-, PC-inhibitable antibodies. A small IgM anti-PC response having a consistent T15-Id+ component is also induced in XID mice, whereas normal mice produce a large IgM response dominated by T15-Id+ antibodies. The immune response to EPC-KLH remains predominantly PC-inhibitable even after multiple immunizations, while the response to DPPC-KLH becomes dominated by PPC-specific antibodies. C.CBA/N mice immunized twice with EPC-KLH are protected against 10(4) S. pneumoniae while as few as 10 bacteria are 100% lethal for the unimmunized controls. The ability of EPC-protein to induce a long-lived, PC-specific response should make this hapten a potential TD vaccine candidate for S. pneumoniae.

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays.

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.