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Huang, C. M., Parsons, M., Wakeland, E. K., Moriwaki, K. and Herzenberg, L. A. (1982). “New immunoglobulin IgG allotypes and haplotypes found in wild mice with monoclonal anti-allotope antibodies.” Journal of Immunology 128(2): 661-7.
Sera from 156 wild mice (Mus musculus L.) collected from parts of Eurasia, Northern Africa, and the Americas were tested in solid-phase radioimmunoassays for reactivity with 20 monoclonal anti-allotope antibodies directed against gene products of the Igh-1, Igh-3, and Igh-4 loci. Most of the wild mice have Igh phenotypes similar to those of inbred strains or heterozygotes thereof, but frequently the wild mice showed unique combinations of allotypes on a given chromosome (haplotypes) not seen in inbred strains. We found new allotypes of the Igh-1 and Igh-4 loci. Mice from three regions (Poland, Taiwan, and Japan) possess combinations of allotypes indicating that recombination, gene conversion, or other gene duplication events have occurred in portions of the Igh constant region gene complex. Most interestingly, sera of three mice from Egypt possess immunoglobulin molecules appearing to result from an intragenic recombination event involving either Igh-1d or Igh-3d with Igh-1b. Two mice from Pakistan and one from Seychelles Island in the Indian Ocean also showed similar unusual phenotypes. These mice possess two CH2 IgH-1b and two CH2 Igh-1a/CH2 Igh-1d determinants, suggesting that these variant immunoglobulin arose from recombinations within the CH2 domain.
Frankel, A. E., Rouse, R. V. and Herzenberg, L. A. (1982). “Human prostate specific and shared differentiation antigens defined by monoclonal antibodies.” Proceedings of the National Academy of Sciences of the United States of America 79(3): 903-7.
Splenic lymphocytes of BALB/c mice immunized with membrane-enriched fractions of human benign prostatic hyperplasia tissues were fused with the NS-1 light chain-secreting murine myeloma cell line. This generated hybridoma cultures that secreted immunoglobulins reactive in solid-phase radioimmunoassays with membrane preparations of prostatic tissues but not with membrane preparations of apparently normal human liver, spleen, thymus, or erythrocytes. After further screening of immunoglobulin reactivities and cloning of cultures, eight monoclonal antibodies were chosen that demonstrated reactivity with human prostate tissues. These monoclonal antibodies could be placed into at least three major groups--epithelium-specific, polyepithelial, and stroma-specific--on the basis of differential binding to the surfaces of various component cells in the prostate and other epithelia. Two antibodies defined unique protein antigens specific for prostate epithelia that were not crossreactive with prostatic acid phosphatase or the recently described "prostatic antigens." These antibodies also detected antigens on malignant prostate tissues as well as other malignant tissues. Four antibodies defined three unique polyepithelial protein antigens (two of the antibodies were different isotypes defining the same protein). Each of the polyepithelial antigens was expressed on a different spectrum of normal epithelial tissues. Two displayed brain tissue crossreactivity, one was present on pancreas, and one was present on platelets. The two antibodies that detected prostatic stromal protein antigens showed different spectra of reactivities. One antibody reacted with apparently all prostatic stromal cells as well as endothelial cells in the prostate and other organs. The other antibody apparently reacted with all prostatic stromal cells as well as myoepithelial and muscle cells in other organs.
Herzenberg, L. A. and Tokuhisa, T. (1982). “Epitope-specific regulation. I. Carrier-specific induction of suppression for IgG anti-hapten antibody responses.” Journal of Experimental Medicine 155(6): 1730-40.
Herzenberg, L. A., Tokuhisa, T. and Parks, D. R. (1982). “Epitope-specific regulation. II. A bistable, Igh-restricted regulatory mechanism central to immunologic memory.” Journal of Experimental Medicine 155(6): 1741-53.
Herzenberg, L. A. and Tokuhisa, T. (1982). “Epitope-specific regulation. III. Induction of allotype-restricted suppression for IgG antibody responses to individual epitopes on complex antigens.” European Journal of Immunology 12(10): 814-8.
Nakai, S., Oi, V., T. and Herzenberg, L. A. (1982). “Nucleotide sequence encoding membrane domains are conserved among immunoglobulin gamma subclass genes.” Biomedical Research 3(1): 37-45.
Dangl, J. L., Parks, D. R., Oi, V. T. and Herzenberg, L. A. (1982). “Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting.” Cytometry 2(6): 395-401.
We have used highly specific, directly fluorescein-conjugated heterologous (conventional) and monoclonal antibodies directed against mouse immunoglobulin isotypes in conjunction with the fluorescence activated cell sorter (FACS) to enrich and clone hybridoma cells producing new immunoglobulin heavy chain constant regions. Each variant retains the parental heavy chain variable region and the parental immunoglobulin light chain; thereby each variant binds the same dansyl (DNS) hapten. These isotype switch variants occur at frequencies of approximately 10-5 to 10-6. We were able to isolate the variants by first sorting for an approximate 1000-fold enrichment of the desired immunoglobulin-producing cells, growing these cells for five to nine days, followed by a second 1000-fold enrichment and direct cell cloning into 96 well culture trays. Clones were screened only 3-5 weeks after the original selection for secretion of dansyl-binding immunoglobulin of the selected isotype. Judicious combination of existing methods permits improved analytical techniques using the cell sorter. These include: first, "red" fluorescence staining of dead cells with ethidium bromide or propidium iodide and using the red fluorescence measurement to exclude dead cells from the green fluorescence selection; and second, the use logarithmic amplification of fluorescence signals, allowing for more succinct selection of fluorescence parameters for sorting.
Herzenberg, L. A., Tokuhisa, T. and Hayakawa, K. (1982). “Lack of immune response gene control for induction of epitope-specific suppression by TGAL antigen.” Nature 295(5847): 329-31.
LAH #208
Frankel, A. E., Rouse, R. V., Wang, M. C., Chu, T. M. and Herzenberg, L. A. (1982). “Monoclonal antibodies to a human prostate antigen.” Cancer Research 42(9): 3714-8.
Three monoclonal antibodies reactive with a purified extractable Mr 34,000 prostate antigen (PA) have been prepared by fusing splenocytes of BALB/c mice preimmunized with purified PA with the NS1 mouse myeloma cell line. The three antibodies were all of the IgG-1 subclass. The antibodies defined two noncross-blocking unique determinants on PA; each present as one site per molecule. IF3 defined one antigenic site and 2G7 and 1C5 defined another antigenic determinant. All of the antibodies reacted with PA in a solid-phase radioimmunoassay and immunoprecipitated 125I-labeled PA. Absorption and sandwich radioimmunoassays showed PA in prostate tissues but not in tonsil, liver, or kidney. Immunoperoxidase staining of formalin-fixed paraffin-embedded sections of benign prostatic hyperplasia and prostatic carcinoma revealed strong prostate epithelial reactivity. None of the antibodies showed reactivity with prostate membrane preparations. A sandwich radioimmunoassay used 2G7 as a plate coat. 125I-labeled 1F3 was used to detect 5 ng PA per ml in sera of patients with prostate cancer. These results confirm previous observations regarding the specificity of PA and shed new evidence for its intracellular localization.
Hardy, R. R., Hayakawa, K., Haaijman, J. and Herzenberg, L. A. (1982). “B-cell subpopulations identified by two-colour fluorescence analysis.” Nature 297(5867): 589-91.
LAH #210
Herzenberg, L. A. (1983). “Allotype suppression and epitope-specific regulation.” Immunol Today 4(4): 113-117.
In neonatal (AxB)F1 mice, injections of maternal (A strain) antibody to the Ig allotype of the paternal (B) strain chromically suppress the production of antibodies wiht the B strain allotype. Here Leonore Herzenberg describes how, in such animals, this form of suppression influences the control of antibody responses to a thymus-dependent antigen that is subsequently encountered.
Dangl, J. L. and Herzenberg, L. A. (1982). “Selection of hybridomas and hybridoma variants using the fluorescence activated cell sorter.” Journal of Immunological Methods 52(1): 1-14.
Parks, D. R., Hardy, Richard A., Herzenberg L.A. (1983). “Dual Immunofluorescence - New Frontiers in Cell Analysis and Sorting.” Immunol. Today 4(5): 145-150.
Hardy, R. R., Hayakawa, K., Haaijman, J. and Herzenberg, L. A. (1982). “B-cell subpopulations identifiable by two-color fluorescence analysis using a dual-laser FACS.” Annals of the New York Academy of Sciences 399: 112-21.
LAH #217
Herzenberg, L. A. (1982). “Epitope-specific regulation of memory B-cell expression.” Annals of the New York Academy of Sciences 399: 368-74.
Herzenberg, L. A., Hayakawa, K., Hardy, R. R., Tokuhisa, T. and Oi, V. T. (1982). “Molecular, cellular and systemic mechanisms for regulating IgCH expression.” Immunological Reviews 67: 5-31.
Matossian-Rogers, A., Rogers, P. and Herzenberg, L. A. (1982). “Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers, Thy-1, Lyt-1, and Lyt-2.” Cellular Immunology 69(1): 91-100.
Matossian-Rogers, A., Rogers, P., Ledbetter, J. A. and Herzenberg, L. A. (1982). “Molecular weight determination of two genetically linked cell surface murine antigens: ThB and Ly-6.” Immunogenetics 15(6): 591-9.
Various murine tumor lines were screened by FACS analysis for the surface antigens ThB and Ly-6.2. Positive cell lines were used for immunoprecipitation studies. A monoclonal ThB-specific antibody immunoprecipitated a unique acidic protein of approximately 16 000 daltons from several positive tumors and from concanavalin A (Con-A) and LPS activated splenic lymphocytes. Monoclonal Ly-6.2-specific antibody was used to immunoprecipitate a 33 500 dalton protein that was shown to exist in four similarly sized forms with different basic charges. In the course of these studies, the apparent molecular weight of the surface antigen T 30, immunoprecipitated with a monoclonal T 30-specific antibody from the cell line EL4, was found to be approximately 25 000 daltons.
Kavathas, P. and Herzenberg, L. A. (1983). “Amplification of a gene coding for human T-cell differentiation antigen.” Nature 306(5941): 385-7.
Using previously isolated mouse L-cell transferents for the human T-cell differentiation antigen Leu-2, we now report the first example of spontaneous gene amplification for membrane antigens. The Leu-2 (or T8) antigen is normally expressed on T lymphocytes that have cytotoxic or suppressor functions. Cells of a Leu-2 transfected clone were stained with fluorescein-tagged monoclonal anti-Leu-2, and the brightest 0.1-0.3% of cells were viably separated using a fluorescence activated cell sorter (FACS). After growth of these selected cells, sorting and regrowth was repeated six times, resulting in a population of cells that, compared with the starting population, stains 40 times brighter for Leu-2 and whose DNA transforms 20 times more efficiently for Leu-2. In addition, these cells have 10- to 50-fold amplified human DNA sequences and numerous double minute chromosome fragments, a common indicator of gene amplication in mouse cells.
LAH #223
Hensleigh, P. A., Herzenberg, L. A., Lipman, S. H., Malvehy, R. M., Medearis, A. L., Moore, M. H., Sutherland, K. K. and Waters, V. B. (1983). “Transient immunologic effects of betamethasone in human pregnancy after suppression of preterm labor.” American Journal of Reproductive Immunology 4(2): 83-7.
Maternal immune suppression is a potentially significant adverse effect when betamethasone is used to hasten lung maturation of the fetus at risk for preterm delivery. However, increased incidence of infection has not been observed consistently after betamethasone treatment of pregnant women. This study was designed to determine if the cellular immune response to steroids may be modified during pregnancy in a way that would diminish the infection risk associated with steroid treatment. The effect of betamethasone on immunocytes among patients with preterm labor or in nonpregnant subjects were determined following administration of 12 mg of betamethasone intramuscularly. We measured serially the circulating leukocytes, lymphocytes, T lymphocytes, and their subsets. Measurements were also made of localized leukocyte mobilization to serum-filled skin chambers covering experimental inflammatory sites. Patients in preterm labor had increased WBC counts prior to treatment with betamethasone but no additional leukocytosis was induced nor was mobilization of leukocytes to the skin chambers decreased. Lymphopenia and depression of T cells was more transient among pregnant patients compared to nonpregnant. Thus, the pregnant patients studied had diminished or more transient potentially adverse immunocyte responses to betamethasone as compared to nonpregnant subjects.
Kavathas, P. and Herzenberg, L. A. (1983). “Stable transformation of mouse L cells for human membrane T-cell differentiation antigens, HLA and beta 2-microglobulin: selection by fluorescence-activated cell sorting.” Proceedings of the National Academy of Sciences of the United States of America 80(2): 524-8.
We isolated stable transformants of mouse L cells expressing human cell surface differentiation antigens by using immunofluorescence with monoclonal antibodies and selection with a fluorescence-activated cell sorter (FACS). Mouse L cells (TK-) were cotransformed with human cellular DNA and the herpes simplex virus thymidine kinase (TK) gene. TK+ transformants were first selected. The TK+ populations were stained with various fluorescent antibodies to membrane antigens, and positive cells were sorted and cloned by using a FACS. Transformants for HLA class I antigens, for beta 2-microglobulin, and for the T-cell differentiation antigens Leu-1 and Leu-2 were isolated. The frequency of antigen transformants among the TK+ transformants was about 0.5 X 10(-3). The sizes of the HLA, Leu-1, and Leu-2 molecules expressed by the transformants were the same as those of the proteins present on DNA-donor cells.
Herzenberg, L. A., K. Hayakawa, R.R. Hardy (1982). “2 Color Facs subpopulation of B Lymphocytes.” Medical Tribune 2: 29-32.
Parsons, M., Oi, V. T., Huang, C. M. and Herzenberg, L. A. (1983). “Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies.” Immunogenetics 18(4): 323-34.
We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1a/Igh-4a heavy chain molecules. The Igh-1a determinants were maintained, but the Igh-4a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.
Medearis, A. L., Hensleigh, P. A., Parks, D. R. and Herzenberg, L. A. (1984). “Detection of fetal erythrocytes in maternal blood post partum with the fluorescence-activated cell sorter.” American Journal of Obstetrics & Gynecology 148(3): 290-5.
A study was made of the frequency and amount of fetal hemorrhage into maternal blood during labor and delivery as evidenced by the number of fetal cells present in the maternal circulation immediately after spontaneous vaginal delivery. A sensitive, indirect immunofluorescence was used with fluorescence-activated cell sorter analysis of erythrocytes. All of the 16 Rh-negative mothers studied after vaginal delivery of Rh-positive infants had circulating Rh-positive cells. The mean Rh-positive to Rh-negative erythrocyte ratio was 1:14, 100 in maternal blood, which corresponds to a mean fetal hemorrhage of 156 microliters. The test described is sufficiently sensitive to be used for the study of primary Rh isoimmunization and could be clinically applicable for antepartum screening to determine which patients require Rh immune globulin treatment before delivery.
Hayakawa, K., Hardy, R. R., Parks, D. R. and Herzenberg, L. A. (1983). “The "Ly-1 B" cell subpopulation in normal immunodefective, and autoimmune mice.” Journal of Experimental Medicine 157(1): 202-18.
A small subpopulation of normal murine splenic B cells carrying all of the classic B cells markers (IgM, IgD, Ia, and ThB) also carries Ly-1, one of the major T cell surface molecules. This "Ly-1 B" subpopulation (identified and characterized by multiparameter FACS analyses) consists of relatively large, high IgM/low-IgD/low-Ly-1 lymphocytes that represent approximately 2% of the spleen cells in normal animals and, generally, 5-10% of spleen cells in NZB mice. Ly-1 B are clearly detectable in all normal mouse strains tested as well as NZB, CBA/N, other X-id mice and nude (nu/nu) mice. They are found primarily in the spleen; are either absent or very poorly represented in lymph node, bone marrow, and thymus; appear early during ontogeny, and comprise about a third of the small number of lymphocytes present in 5-d-old mice. NZB and (NZB x NZW)F1 mice have more Ly-1 B than all other strains and, furthermore, have a unique Ly-1 B population that secretes IgM when cultured under usual conditions in the absence of added antigen. The IgM secretion by these Ly-1 B cells accounts for the previously reported "spontaneous" IgM secretion by NZB spleen cells in culture. Studies with FACS-sorted cells show that the presence of Ly-1 on these IgM-secreting cells distinguishes them from the (Ly-1 negative) IgM-secreting "direct" plaque-forming cells generated in NZB mice after stimulation with sheep erythrocytes.
Hensleigh, P. A., Waters, V. B. and Herzenberg, L. A. (1983). “Human T lymphocyte differentiation antigens: effects of blood sample storage on Leu antibody binding.” Cytometry 3(6): 453-5.
Current studies of human T lymphocytes and their subsets often use quantitative immunofluorescence analysis with monoclonal antibodies against cell surface antigens. With storage of whole blood or separated mononuclear cells for more than a few hours we have found marked changes in lymphocyte analysis using a fluorescence activated cell sorter (FACS). Experiments were done to determine if these lymphocyte changes were influenced by storage temperature and if lymphocytes could be made more stable by addition of culture media RPMI 1640 to whole blood. Optimal conditions found for blood storage were with with addition of 50% RPMI 1640 and with samples held at room temperature (22 degrees C). With these storage conditions, delay on FACS analysis up to 24 hours did not result in spurious results. When blood samples are collected in places remote from the laboratory or when batch analysis of serially collected samples is desirable, excessive storage times should be avoided.
Zelaschi, D., Newby, C., Parsons, M., van West, B., Cavalli-Sforza, L. L. and Herzenberg, L. A. (1983). “Human immunoglobulin allotypes: previously unrecognized determinants and alleles defined with monoclonal antibodies.” Proceedings of the National Academy of Sciences of the United States of America 80(12): 3762-6.
The highly polymorphic system of serologically defined genetic markers on human IgG heavy chains (Gm allotypes) is second only to the HLA complex in terms of the large number of determinants, alleles, and haplotypes that can be used for analyses of disease associations and other genetic studies. However, present typing methods are based on the use of anti-Gm antisera that are derived mainly from fortuitously immunized human donors, often requiring processing before use, and must be used in a hemagglutination-inhibition assay that cannot be used in typing for isoallotypic determinants (currently termed "non-markers"). In studies presented here, we describe an allotyping system that utilizes monoclonal antibodies in a "sandwich" modification of the solid-phase radioimmunoassay, which is capable of reliable quantitative typing of allotypic, isoallotypic, and isotypic immunoglobulin determinants. We show that these highly reproducible, easily disseminated, and essentially inexhaustible reagents can be used for rapid, sensitive, and quantitative Gm typing. Using this system we define two previously unrecognized Gm determinants, one of which, found to date only in Caucasians, is different from all known Gm markers and thus defines previously unrecognized alleles and haplotypes. The other determinant co-segregates with the conventional G3m(b1) marker but is distinct from that marker on serological grounds. The successful preparation of mouse monoclonal antibodies that detect human Gm allotypic differences and the development of an assay system capable of typing isoallotypic as well as allotypic determinants opens the way to further dissection and application of this rich genetic system.
Oi, V. T., Morrison, S. L., Herzenberg, L. A. and Berg, P. (1983). “Immunoglobulin gene expression in transformed lymphoid cells.” Proceedings of the National Academy of Sciences of the United States of America 80(3): 825-9.
Myeloma, hybridoma, and thymoma cell lines have been successfully transfected for the Escherichia coli xanthine-guanine phosphoribosyltransferase gene (gpt) by using the plasmid vector pSV2-gpt. The transformed cells synthesize the bacterial enzyme 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase (XGPRT; EC 2.4.2.22) and have been maintained in selective medium for over 4 months. Lymphoid cell lines expressing a K immunoglobulin light chain were obtained by transfecting cells with pSV2-gpt containing a rearranged K light chain genomic segment from the S107 myeloma cell line. The S107 light chain is synthesized in gpt-transformed J558L myeloma cells and is identical to the light chain synthesized by the S107 myeloma cell line, as judged by immunoprecipitation and two-dimensional gel electrophoresis. Furthermore, this light chain is synthesized and secreted as part of an intact antibody molecule by transformed hybridoma cells that normally secrete an IgGl (gamma, K) antibody molecule. No light chain synthesis was detected in a similarly transformed rat myeloma or a mouse thymoma line.
Herzenberg, L. A., Tokuhisa, T. and Hayakawa, K. (1983). “Epitope-specific regulation.” Annual Review of Immunology 1: 609-32.
LAH #233
Haaijman, J. J., Hardy, R. R., Hayakawa, K. and Herzenberg, L. A. (1982). “Distinct B Cell Subpopulations in Mice as characterized by multiparameter fluorescence activated cell sorter (FACS) analysis.” Journal of Experimental Medicine Submitted.
Hardy, R. R., Hayakawa, K., Parks, D. R. and Herzenberg, L. A. (1983). “Demonstration of B-cell maturation in X-linked immunodeficient mice by simultaneous three-colour immunofluorescence.” Nature 306(5940): 270-2.
CBA/N mice carrying the X-linked immune deficiency gene (xid) have fewer splenic B cells than normal CBA mice and are unresponsive to a certain class of antigens. Studies of B-cell surface-marker expression and immune responsiveness have led to the commonly accepted idea that the B cells in adult xid mice are immature and resemble the B cells of young (1-3 week old) normal mice. That is, like young animals, xid mice lack cells in the most numerous of three IgM/IgD B-cell subpopulations (designated I in Fig. 1a, b) present in adult spleen. We now report, however, that this picture is an oversimplification and that in fact the B cells in adult xid mice differ from those present in either adult or young normal mice. Using quantitative three-colour fluorescence-activated cell sorter (FACS) analyses, we have compared the correlated expression of IgM, IgD and a newly discovered B-lymphocyte antigen (BLA-1) on splenic B cells in normal and xid mice. We show here (1) that most B cells in adult xid mice (as in normals) are BLA-1- whereas all B cells in young animals are BLA-1+; (2) that the major difference in the IgM/IgD B-cell subpopulations found between xid and normal mice is limited to the BLA-1- cells; and (3) that xid mice have increased numbers of BLA-1+ population III B cells.
Schroder, J., Nikinmaa, B., Kavathas, P. and Herzenberg, L. A. (1983). “Fluorescence-activated cell sorting of mouse-human hybrid cells aids in locating the gene for the Leu 7 (HNK-1) antigen to human chromosome 11.” Proceedings of the National Academy of Sciences of the United States of America 80(11): 3421-4.
Leu 7 (HNK-1) is a membrane antigen expressed on human natural killer cells and some other lymphoid cells. Starting with two clones of mouse-human hybrid lymphoid cells that had 1.6% and 35% Leu 7-positive cells, respectively, we viably sorted Leu 7-positive and -negative cells using a fluorescence-activated cell sorter (FACS). Short-term progeny of the sorted cells were then karyotyped. Chromosome 11 was the only human chromosome that was absent from the Leu 7-negative population and present in nearly all of the progeny of the Leu 7-positive selected cells. Thus, we assigned the Leu 7 gene to chromosome 11.
Huang, C. M., Parsons, M., Oi, V. T., Huang, H. J. and Herzenberg, L. A. (1983). “Genetic characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies.” Immunogenetics 18(4): 311-21.
We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG1, IgG2a, and IgG2b. In this communication we describe their reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Ighp.
Lanier, L. L., Engleman, E. G., Gatenby, P., Babcock, G. F., Warner, N. L. and Herzenberg, L. A. (1983). “Correlation of functional properties of human lymphoid cell subsets and surface marker phenotypes using multiparameter analysis and flow cytometry.” Immunological Reviews 74: 143-60.
Oi, V. T., Herzenberg, L. A. and Birshtein, B. K. (1983). “Localization of murine Igh-1a allotypic determinants by using a panel of mouse myeloma variant immunoglobulins.” Journal of Immunology 130(4): 1967-9.
A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobulin allotypic determinants. By determining which ones are present on a panel of hybrid IgG2b-IgG2a immunoglobulins, we have localized some of the allotypic determinants present on the IgG2a heavy chain of the "a" allotype (Igh-1a proteins). In particular, one group of determinants--Ig(1a)9.8 (20.6B8), 17.2 (20.19.2), and 14.4 (21.74.4)--has been placed in the CH2 domain. A second group--Ig(1a)8.3 (20.8.3), 21.2 (20.11.2), and 15.3 (21.66.3)--is located in a segment spanning the C terminal 8 residues of the CH2 domain and the complete CH3 domain.
Parham, P., Kipps, T. J., Ward, F. E. and Herzenberg, L. A. (1983). “Isolation of heavy chain class switch variants of a monoclonal anti-DC1 hybridoma cell line: effective conversion of noncytotoxic IgG1 antibodies to cytotoxic IgG2 antibodies.” Human Immunology 8(2): 141-51.
Spontaneously arising class switch variants of the Genox 3.53 hybridoma cell line were isolated. They secrete IgG2a or IgG2b monoclonal antibodies of anti-DC1 specificity identical to that of the IgG1 secreting parental cell. In contrast to the parental monoclonal antibody, those secreted by the variants are cytotoxic to peripheral blood B lymphocytes of DC1 positive individuals and are thus compatible with existing HLA typing techniques. This provides a general method for converting noncytotoxic anti-HLA antibodies into cytotoxic typing reagents.
Oi, V. T., Vuong, T. M., Hardy, R., Reidler, J., Dangle, J., Herzenberg, L. A. and Stryer, L. (1984). “Correlation between segmental flexibility and effector function of antibodies.” Nature 307(5947): 136-40.
Mouse monoclonal anti-dansyl antibodies with the same antigen-binding sites but different heavy chain constant regions were generated. The extent of segmental flexibility in times of nanoseconds and the capacity to fix complement were greatest for IgG2b, intermediate for IgG2a, and least for IgG1 and IgE. Hence, the effector functions of immunoglobulin isotypes may be controlled in part by the freedom of movement of their Fab arms.
Hayakawa, K., Hardy, R. R., Herzenberg, L. A., Steinberg, A. D. and Herzenberg, L. A. (1984). ly-1 B: Afunctionally distinct B-cell subpopulation. Progress of Immunology V. Tokyo, Academic Press. V: 661-668.
Alberti, S., Stovel, R. and Herzenberg, L. A. (1984). “Preservation of cells sorted individually onto microscope slides with a fluorescence-activated cell sorter.” Cytometry 5(6): 644-7.
Fluorescence-activated cell sorters permit analyses and separation of cell populations based on light scatter and surface immunofluorescence parameters. Since a sorter can deposit individually identifiable cells onto a microscope slide, it was considered of interest to combine the flow measurements with analyses available on cells adhering to a surface as in, for example, morphological studies, cytoplasmic immunofluorescent staining, and mRNA in situ hybridization. A necessary condition for these studies is the preservation of cell structures after sorting. We report here a procedure suitable for this purpose. The most important features of this procedure are A) reducing the saline content of the sorter sheath fluid to about 0.0015 M (one-hundredth that of normal saline) to prevent cell damage due to hypertonicity during drying, and B) coating the substrate with a thin layer of newborn calf serum to promote the adherence of the cells to the substrate during subsequent fixing and staining.
Waldor, M. K., Hardy, R. R., Hayakawa, K., Steinman, L. and Herzenberg, L. A. (1984). “Disappearance and reappearance of B cells after in vivo treatment with monoclonal anti-I-A antibodies.” Proceedings of the National Academy of Sciences of the United States of America 81(9): 2855-8.
Previous studies have shown that treatment with antibodies to the murine I-A antigen encoded in the major histocompatibility complex attenuates experimental allergic encephalitis and experimental autoimmune myasthenia gravis. These studies were conducted with SJL mice, an inbred strain that is highly susceptible to the induction of these diseases. Here we show that injection of monoclonal anti-I-A antibody in the amounts used for the above studies rapidly depletes B cells. Fluorescence-activated cell sorter (FACS) multiparameter analysis of the B-cell subpopulations in treated animals shows that maximum depletion occurs around 5 days after treatment and that recovery of some subpopulations i still incomplete 1 month later. SJL mice are more sensitive to this B-cell depletion and recover more slowly than putatively normal C3H.Ighb (CKB) mice. Some components of the primary, secondary and tertiary IgG antibody responses are reduced in anti-I-A-treated SJL animals immunized after the first and second anti-I-A injections. The persistence of some antibody response impairment well beyond the time when anti-I-A disappears raises a note of caution concerning human therapy protocols based on the injection of anti-Ia antibodies.
Herzenberg, L. A. (1983). Epitope-Specific Regulation: A Brief Overview. Progress in Immunology V. Japan, Academic Press Japan, Inc. V: 581-589.
Parks, D. R., Hardy, R. R. and Herzenberg, L. A. (1984). “Three-color immunofluorescence analysis of mouse B-lymphocyte subpopulations.” Cytometry 5(2): 159-68.
We have modified a fluorescence-activated cell sorter (FACS) to make three independent immunofluorescence measurements on each cell and used this system to study mouse B-lymphocyte subpopulations. An argon-ion laser (emitting at 488 nm) excites fluorescein- and phycoerythrin-labeled reagents, and a tunable dye laser charged with rhodamine 6G (emitting at 615 nm) excites an allophycocyanin-labeled reagent. We report simultaneous measurements of IgM, IgD, and the recently-defined mouse B lymphocyte antigens BLA-1 and BLA-2 on splenic lymphocytes of CBA/J mice and mice of the congenic strain CBA/N (which have an X-linked immunodeficiency [xid]). These data provide information on relationships among the B-cell populations in CBA/J "normal" mice and the defective CBA/N that could not be derived from one- or two-color immunofluorescent measurements. We believe this is the first use of allophycocyanin as an immunofluorescence label.
LAH #246-01
Parks, D. R. and Herzenberg, L. A. (1984). “Fluorescence-activated cell sorting: theory, experimental optimization, and applications in lymphoid cell biology.” Methods in Enzymology 108: 197-241.
Herzenberg, L. A., P. Kavathis (1984). Transformation and amplification of genes for human differential antigens in mouse fibroblasts,. Immunogenetics: Its Application to Clinical Medicine. Tokyo, Academic Press Japan, Inc.: 283-284.
Hsu, C., Kavathas, P. and Herzenberg, L. A. (1984). “Cell-surface antigens expressed on L-cells transfected with whole DNA from non-expressing and expressing cells.” Nature 312(5989): 68-9.
We have shown previously that transfection of mouse L-cells with DNA from JM, a human T-cell line expressing certain T-cell differentiation antigens, yields stable transfectants expressing one or another of these antigens. The identities of the antigens were confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. We now report that our procedure--co-transfection with the chicken thymidine kinase gene (tk) and whole cellular DNA, selection with hypoxanthine-aminopterin-thymidine (HAT), and staining of the cells with fluorochrome-conjugated monoclonal antibodies and fluorescence-activated cell-sorter (FACS) selection--yields transfectants expressing a variety of cell-surface molecules (19 of 21 investigated), most at a frequency of about one per 10(3) Tk+ transformants. Of these, 9 of 12 were transferred and expressed as readily using DNA from cells which did not express the cell-surface antigens as from tissues or cells that did express them.
Kawata, M., Sizer, K., Sekiya, S., Parnes, J. R. and Herzenberg, L. A. (1984). “Limitation of differential expression of HLA-A,B,C antigens on choriocarcinoma cell lines by messenger RNA for HLA heavy chain but not by beta 2-microglobulin.” Cancer Research 44(9): 4011-6.
The relative amounts of HLA-A,B,C antigens, beta 2-microglobulin (beta 2m), and trophoblast antigens (Trop-1 and Trop-2) were determined on nine choriocarcinoma cell lines including seven lines of gestational origin and two lines of nongestational origin (from ovary and stomach) by quantitative immunofluorescence analysis using a fluorescence-activated cell sorter. Most of these lines expressed surface HLA to variable extents, but one had none detectable. However, all lines secreted readily measurable amounts of beta 2m. We analyzed total RNA extracted from these lines using northern blot molecular hybridization with HLA-A,B,C- and beta 2m-specific complementary DNA probes. We found no messenger RNA species which hybridized with the HLA probe in cells with no detectable HLA surface antigen and only small amounts of HLA-specific RNA in cells with low levels of HLA membrane antigen. Cells exhibiting surface HLA levels greater than about 30% of that on lymphocytes had much higher amounts of HLA-specific RNA than did choriocarcinoma cells with no or low HLA antigen expression. In contrast, RNA hybridizing with beta 2m-specific probes was present at the 20% level or higher (relative to lymphocytes) in all the cell lines tested. Thus, the expression of HLA-A,B,C is apparently limited in choriocarcinoma cells by the level of HLA heavy-chain RNA and not by the level of beta 2m RNA. We discuss these findings in relation to the normal trophoblastic or other origins of this tumor type and with respect to the regulation and function of HLA in trophoblasts.
Kawata, M., Parnes, J. R. and Herzenberg, L. A. (1984). “Transcriptional control of HLA-A,B,C antigen in human placental cytotrophoblast isolated using trophoblast- and HLA-specific monoclonal antibodies and the fluorescence-activated cell sorter.” Journal of Experimental Medicine 160(3): 633-51.
Human placental cell suspensions prepared by trypsin digestion were analyzed with several monoclonal antibodies on a multiparameter fluorescence-activated cell sorter (FACS). Five distinct cell populations were isolated on the basis of size and quantitative differences in the coordinate expression of cell surface antigens detected by monoclonal antibodies against an HLA-A,B,C monomorphic determinant (MB40.5) and against human trophoblasts (anti-Trop-2). By FACS analysis and after sorting we clearly identified the major cell population as cytotrophoblasts based on several independent criteria, including presence of trophoblast-specific surface antigens, Trop-1, and Trop-2; absence of all HLA class I, class II, and beta 2-microglobulin (beta 2m) antigens; absence of the pan-leucocyte and monocyte antigens, HLe1 and LeuM1, respectively; presence of Y-chromatin in a male placenta; presence of placental and not liver alkaline phosphatase; and a large, mononuclear morphology. These procedures provide a reproducible method for obtaining highly purified human cytotrophoblast populations for further studies. We measured by molecular hybridization (RNA or Northern blots) the HLA-A,B,C and beta 2m mRNA in total RNA extracted from sorted cytotrophoblasts. We find that normal human cytotrophoblasts have extremely small amounts of HLA-A,B,C mRNA: approximately 300 times less than that in the lymphoid cell line LCL-721 or normal lymphocytes. In contrast, they have approximately 11% the level of beta 2m mRNA present in LCL-721 cells. Thus, HLA-A,B,C antigen expression on human cytotrophoblasts is limited by the level of HLA heavy chain mRNA.
Hardy, R. R., Hayakawa, K., Parks, D. R. and Herzenberg, L. A. (1984). “Murine B cell differentiation lineages.” Journal of Experimental Medicine 159(4): 1169-88.
Subpopulations of mouse B cells express different amounts of two antigens (BLA-1 and BLA-2) recognized by rat monoclonal antibodies (53-10.1 and 30-E2). Two-color immunofluorescence analysis on the fluorescence-activated cell sorter (FACS) shows that the 53-10.1 monoclonal antibody reacts with a similar proportion of splenic B cells from normal and CBA/N (xid) mice, whereas 30-E2 reacts with most CBA/N B cells but with only a fraction of normal B cells. Data from three- and four-color immunofluorescence analyses with xid, athymic (nude), and normal mice suggest that the order in which these antigens are lost during B cell differentiation distinguishes two B cell lineages: immature B cells express both antigens, intermediate-stage B cells of one or the other lineage express only BLA-1 or only BLA-2, respectively, and mature resting B cells express neither. CBA/N mice lack one of the putative intermediate populations (BLA-1+,2-); thus, this population apparently gives rise to the predominant mature B cell population, which is present in normal adult spleen and lymph node but is missing in CBA/N. The other putative intermediate population (BLA-1-,2+) is decreased by two- to threefold in spleens from nude mice compared with strain-matched controls. Both BLA-1 and BLA-2 antigens rapidly reappear after specific (antigen) or nonspecific (lipopolysaccharide) B cell activation. IgM plaque-forming cells (PFC) derived from such activated cells continue to express both antigens while IgG PFC express only BLA-1.
Hayakawa, K., Hardy, R. R., Honda, M., Herzenberg, L. A. and Steinberg, A. D. (1984). “Ly-1 B cells: functionally distinct lymphocytes that secrete IgM autoantibodies.” Proceedings of the National Academy of Sciences of the United States of America 81(8): 2494-8.
Studies presented here introduce another perspective on the mechanisms responsible for IgM autoantibody production. A unique subpopulation of B lymphocytes (Ly-1 B) that concomitantly expresses IgM, IgD, Ia, and Ly-1 membrane glycoproteins is present at higher frequencies in NZB and NZB-related mice. The Ly-1 B subpopulation in these autoimmune animals is responsible for the "spontaneous" IgM secretion demonstrated with cultured NZB spleen cells and contains the cells that secrete typical NZB IgM autoantibodies to single-stranded DNA and to thymocytes. In addition, the Ly-1 B population in normal mouse strains (and in NZB) contains virtually all of the spleen cells that secrete IgM autoantibodies reactive with bromelain-treated mouse erythrocytes. Since a different B-cell subpopulation (IgM+, IgD-, Ly-1) secretes most of the IgM antibodies produced in responses to exogenous antigens, we conclude that Ly-1 B cells constitute a functionally distinct B-cell population important in certain kinds of autoimmunity.
Oi, V. T., Hsu, C., Hardy, R. R. and Herzenberg, L. A. (1984). Hybridoma Antibody-Producing Switch Variants: A Variant Lacking the CH1 Domain. Cell Fusion: Gene Transfer and Transformation. R. F. B. E. G. Bassett. New Yok, Raven Press: 281-287.
Kipps, T. J. and Herzenberg, L. A. (1986). Hybridoma immunoglobulin isotype switch variant selection using the fluorescence activated cell sorter. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 4; Applications of immunological methods in biomedical sciences: 109.1-109.8.
In 1978, Rajewsky et al. utilized the powerful resolving capacity of the fluorescence activated cell sorter (FACS) to isolate isotype 'switch variant' myeloma cels from a large culture of myeloma cells. Using fluorescence-conjugated antibody specific for an immunoglobulin isotype not expressed by the parent myeloma cell line, they were successful in staining and subsequently sorting rare switch variant cells. These variant cells secreted myeloma protein of a different immunoglobulin isotype but identical variable (V) region idiotype(s) as the antibody produced by the parent myeloma line. As such, these cells apparently switched from the expression of the gene encoding the constant part of one heavy chain to that of another, while expressing the same heavy chain variable region gene which encodes the antibody's antigen combining site. Although other investigators had observed similar isotype switch variants after mutagenesis of the MPC 11 myeloma cell ilne using a soft agar cloning technique, these investigators demonstrated that rare spontaneous switch variant cells could be selectively stained for the variant immunoglobulin expressed on their surface membrane and subsequently sorted using the FACS.
LAH #256
Hardy, R. R., Hayakawa, K., Herzenberg, L. A., Morse, H. C. d. and Davidson, W. F. (1984). “Ly-1 as a differentiation antigen on normal and neoplastic B cells.” Current Topics in Microbiology & Immunology 113: 231-6.
Kavathas, P., Sukhatme, V. P., Herzenberg, L. A. and Parnes, J. R. (1984). “Isolation of the gene encoding the human T-lymphocyte differentiation antigen Leu-2 (T8) by gene transfer and cDNA subtraction.” Proceedings of the National Academy of Sciences of the United States of America 81(24): 7688-92.
We report the isolation of genomic and cDNA clones encoding the human T-lymphocyte cell-surface differentiation antigen, Leu-2 (T8), by use of a combination of transfection, fluorescence-activated cell-sorting, and subtractive cDNA hybridization. We constructed a cDNA library with mRNA from a mouse L-cell transfectant in which the human Leu-2 gene is expressed and amplified. We identified Leu-2 cDNA clones by screening with a selected cDNA probe from a second amplified Leu-2 transfectant. This probe contained cDNA species not removed by hybridization with L-cell mRNA. A Leu-2 cDNA clone was used to isolate a genomic clone. Transfection with DNA from this clone resulted in a high number of Leu-2 transfectants. This approach can be used to clone genes coding for other cell-surface molecules.
Kipps, T. J., and Leonard A. Herzenberg (1986). Schemata for the production of monoclonal antibody producing hybridomas. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 4; Applications of immunological methods in biomedical sciences: 108.1-108.9.
Kavathas, P. a. L. A. H. (1986). Transfection for lymphocyte cell surface antigens. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 3; Genetics and Molecular Immunology: 91.1 - 91.10.
LAH #259-01
Kavathas, P. (1986). Amplification and molecular cloning of transfected genes. Hybridomas in Biotechnology and Medicine. T. A. Springer, Plenum Press. N/A.
Zelaschi, D., M.J. Johnson, C.J. Newby and Leonore A. Herzenberg (1986). New methods for human typing. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 3; Genetics and Molecular Immunology: 95.1 - 95.10.
Hardy, R. R. (1986). Purification and Characterization of Monoclonal Antibodies. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 1; Immunochemistry: 13.1 -.
Hardy, R. R. (1986). Complement fixation by defined antibody-antigen complexes. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 1; Immunochemistry: 40.1 -.
Hardy, R. R. (1986). Purification and coupling of fluorescent proteins for use in flow cytometry. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 1; Immunochemistry: 31.1 - 31.12.
Herzenberg, L. A. (1986). A brief overview: Epitope-specific regulation. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell, L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 2; Cellular Immunology: 74.1 -.
Parsons, M., Leonore A. Herzenbeg, Alan M. Stall and Leonard A. Herzenberg (1986). Mouse immunoglobulin allotypes: description and special methodology. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 3; Genetics and Molecular Immunology: 97.1 - 97.17.
Newby, C. J. and Herzenberg, L. A. H., K. (1986). Solid-phase radio-immunoassaya. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell, and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publication, Ltd. 1; Immunochemistry: 34.1 -.
Parks, D., Lanier, L. and Herzenberg, L. A. (1986). Flow cytometry and fluorescence activated cell sorting. The Handbook of Experimental Immunology. L. A. Herzenberg, D. M. Weir, C. C. Blackwell and L. A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 1 Immunoochemisry: 291-29.21.
Moore, W. and Kautz, R. (1986). Data analysis for flow cytometry. The Handbook of Experimental Immunology. L. A. H. D.M. Weir, C.C. Blackwell, and L.A. Herzenberg. Edinburgh, Blackwell Scientific Publications, Ltd. 1; Immunochemistry: 30.1 -.
Kipps, T. J. and Herzenberg, L. A. (1985). Fluorescence activated cell sorter and monoclonal antibodies: complementary tools in immunodiagnosis and immuntherapy. Rapid methods and automation in microbiology and immunology. Habermehl. Heidelberg, Springer Verlag. One: 115 - 122.
Herzenberg, L. A., Hsu, C., Alberti, S. and Kavathas, P. (1984). “Transfection and cloning of genes for membrane antigens using the FACS.” Medical Oncology & Tumor Pharmacotherapy 1(4): 219-24.
In order to facilitate cloning of genes for cell surface molecules, we cotransfected LTK- mouse fibroblasts with thymidine kinase (TK) genes and total human or mouse DNA. TK+ cells, selected by growth in HAT medium, were stained with fluorochrome conjugated monoclonal antibodies or other fluorescent ligands which bind to one or another membrane differentiation antigen or receptor. We isolated fluorescent transfectants expressing these molecules using a fluorescence activated cell sorter (FACS). For some antigens, spontaneous gene amplification occurred. By repeated cycles of FACS sorting and regrowth we obtained high expressing clones. We then isolated cDNA and genomic clones using selected cDNA probes to screen phage with cDNA inserts. DNA from virtually any tissue source transfected equally well for the various molecules except for DNA from a trophoblast derived choriocarcinoma cell line which did not transfect for Leu-2.
Morrison, S. L., Johnson, M. J., Herzenberg, L. A. and Oi, V. T. (1984). “Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains.” Proceedings of the National Academy of Sciences of the United States of America 81(21): 6851-5.
We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.
Tagawa, M., Tokuhisa, T., Ono, K., Taniguchi, M. and Herzenberg, L. A. (1984). “Epitope-specific regulation. IV. In vitro studies with suppressor T cells induced by carrier/hapten-carrier immunization.” Cellular Immunology 86(2): 327-36.
Sequential immunization with a carrier molecule and a new epitope (hapten) conjugated to the carrier (carrier/hapten-carrier immunization) induces specific suppression for IgG antibody production to the new epitope (hapten) on the carrier. Once induced, this "epitope-specific" suppression persists and specifically suppresses subsequent in vivo IgG antibody responses to the hapten presented on the same or on an unrelated carrier molecule. In vitro studies presented here characterize the surface markers and specificity of suppressor T cells generated in carrier/hapten-carrier-immunized animals. Thus we show (1) that spleen cells from these donors suppress in vitro IgG anti-hapten antibody production by cocultured hapten-primed spleen cells; (2) that some but not all of the suppressor cells carry surface Lyt-2; (3) that at least some of the suppressor cells have receptors for the inducing hapten (DNP); and (4) that, unlike the suppression obtained in vivo, the in vitro suppression extends to IgG responses to unrelated carrier protein epitopes presented in association with the inducing hapten.
Waldor, M. K., Sriram, S., Hardy, R., Herzenberg, L. A., Lanier, L., Lim, M. and Steinman, L. (1985). “Reversal of experimental allergic encephalomyelitis with monoclonal antibody to a T-cell subset marker.” Science 227(4685): 415-7.
Administration of a monoclonal antibody (GK1.5) that recognizes the L3T4 marker present on helper T cells prevented the development of experimental allergic encephalomyelitis (EAE) in mice. Furthermore, treatment with GK1.5 reversed EAE when the antibody was given to paralyzed animals. In vivo injection of GK1.5 selectively reduced the number of L3T4+ cells in the spleen and the lymph nodes. These results suggest that manipulation of the human equivalent of the murine L3T4+ T-cell subset with monoclonal antibodies may provide effective therapy for certain autoimmune diseases.
Kipps, T. J., Parham, P., Punt, J. and Herzenberg, L. A. (1985). "Importance of Immunolglobulin Isotype in Human Antibody-Dependent, Cell-Mediated Cytotoxicity Directed by Murine Monoclonal Antibodies." Journal of Experimental Medicine 161: 1-17
LAH #272-03
Kipps, T. J. (1985). Switching the isotype of monoclonal antibodies. Hybridoma Technology in theBiosciences and Medicine. T. Springer. New York, Plenum Press. One: 89-101.
Parham, P., Antonelli, P., Herzenberg, L. A., Kipps, T. J., Fuller, A. and Ward, F. E. (1986). “Further studies on the epitopes of HLA-B7 defined by murine monoclonal antibodies.” Human Immunology 15(1): 44-67.
Monoclonal antibodies reactive with polymorphic epitopes of HLA-B7 were analyzed by direct and indirect cytotoxicity assays on established panels of HLA typed lymphocytes. This permitted further refinement of their specificity and the identification of various novel reactions. The topographic relationship of polymorphic epitopes on the surface of the B7 molecule was assessed with various serological assays using cell surface B7 or papain solubilized B7 as the antigenic target. These studies focused on monoclonal antibodies recognizing B27 and B7. The results, in combination with those of previously published studies, are used to provide a current assessment of the epitope map of HLA-B7 as defined with mouse monoclonal antibodies. This is compared to the results obtained with alloantisera.
Suomalainen, H. A., Herzenberg, L. A., Gahmberg, C. G., Sussman, H. H. and Schroder, J. (1985). “Assignment of gene for human cell-surface membrane antigen Trop-4 to chromosome 11.” Somatic Cell & Molecular Genetics 11(3): 257-65.
The gene (named MF16) for a surface membrane antigen, Trop-4, is assigned to human chromosome 11 on the basis of studies using a mouse monoclonal antibody, immunofluorescence, fluorescence-activated cell sorting (FACS), immunoprecipitation, and mouse-human lymphocyte hybrids. The Trop-4 antigen is present on all human cell lines tested, on peripheral blood monocytes and granulocytes, and on a small fraction of peripheral blood lymphocytes, but is absent from erythrocytes. The Trop-4 monoclonal antibody precipitates an 85,000-dalton glycopolypeptide from hybrid cells containing human chromosome 11. However, in a human cell line expressing this antigen, a larger-molecular-weight species, 100-105,000 daltons was coprecipitated with the 85,000-dalton glycopeptide, and under nonreducing conditions a larger compound of 110-125,000 daltons was obtained. Although the Trop-4 antigen is of similar molecular weight to the Mab-4 and F10.44.2 antigens previously assigned to chromosome 11, it is shown to be different from them.
Kipps, T. J. and Herzenberg, L. A. (1986). “Homologous chromosome recombination generating immunoglobulin allotype and isotype switch variants.” EMBO Journal 5(2): 263-8.
We investigated whether spontaneous isotype switching in monoclonal antibody-producing hybridomas always occurs with genes on the same chromosome. Spleen cells of (BAB/ 25 X AKR/J) F1 mice, immunized with dansyl-keyhole limpet hemocyanin (DNS-KLH), were hybridized with NS-1 to generate hybridomas producing monoclonal anti-DNS antibodies of either the b or d haplotype of the BAB/25 or AKR/J parent, respectively. We selected isotype switch variants of such hybridomas using the fluorescence-activated cell sorter (FACS). Although in most cases the allotypic haplotype expressed by the parent and switch-variant hybridomas are the same, in one family of variants we noted a switch in haplotype along with the switch in isotype. This was noted in the selection of IgG2a switch variants from an IgG1 switch variant originally derived from an IgG3-producing parent. Biochemical and molecular studies confirm that the allotype switch variant expresses the same heavy-chain variable region gene complex as its parent hybridomas. As such, the allotype switch represents an example of spontaneous mitotic recombination between immunoglobulin heavy-chain genes, generating a single actively transcribed gene from loci previously positioned on different chromosomes.
Hayakawa, K., Hardy, R. R. and Herzenberg, L. A. (1985). “Progenitors for Ly-1 B cells are distinct from progenitors for other B cells.” Journal of Experimental Medicine 161(6): 1554-68.
Data from previous multiparameter fluorescence-activated cell sorter (FACS) analysis and sorting studies define a subset of murine B cells that expresses the Ly-1 surface determinant in conjunction with IgM, IgD, Ia, and other typical B cell markers. These Ly-1 B cells are physically and functionally distinct. They express more IgM and less IgD than most other B cells; they are not normally found in lymph node or bone marrow; they are always present at low frequencies (1-5%) in normal spleens, and, as we show here, they comprise about half of the B cells (10-20% of total cells) recovered from the peritoneal cavity in normal mice. Furthermore, most of the commonly studied IgM autoantibodies in normal and autoimmune mice are produced by these Ly-1 B cells, even though they seldom produce antibodies to exogenous antigens such as trinitrophenyl-Ficoll or trinitrophenyl-keyhole limpet hemocyanin. Cell transfer studies presented here demonstrate that the progenitors of Ly-1 B cells are different from the progenitors of the predominant B cell populations in spleen and lymph node. In these studies, we used FACS analysis and functional assays to characterize donor-derived (allotype-marked) B cells present in lethally irradiated recipients 1-2 mo after transfer. Surprisingly, adult bone marrow cells typically used to reconstitute B cells in irradiated recipients selectively failed to reconstitute the Ly-1 B subset. Liver, spleen, and bone marrow cells from young mice, in contrast, reconstituted all B cells (including Ly-1 B), and peritoneal "washout" cells (PerC) from adult mice uniquely reconstituted Ly-1 B. Bone marrow did not block Ly-1 B development, since PerC and newborn liver still gave rise to Ly-1 B when jointly transferred with marrow. These findings tentatively assign Ly-1 B to a distinct developmental lineage originating from progenitors that inhabit the same locations as other B cell progenitors in young animals, but move to unique location(s) in adults.
Hardy, R. R., Dangl, J. L., Hayakawa, K., Jager, G. and Herzenberg, L. A. (1986). “Frequent lambda light chain gene rearrangement and expression in a Ly-1 B lymphoma with a productive kappa chain allele.” Proceedings of the National Academy of Sciences of the United States of America 83(5): 1438-42.
We describe here a murine Ly-1-bearing pre-B-cell tumor that, when induced for kappa light chain expression with bacterial lipopolysaccharide, also gives rise spontaneously to a few percent of cells expressing surface lambda light chains. These lambda-positive cells have undergone DNA rearrangements involving either V lambda 1 or V lambda 2 genes. Nearly all clones of lambda-bearing cells express mu and lambda on their surface (but not kappa). However, all these lambda-positive clones continue to transcribe kappa mRNA and synthesize internal kappa chains. Further, surface lambda-positive clones show JH rearrangements on one or both heavy chain chromosomes.
Sidman, C. L., Shultz, L. D., Hardy, R. R., Hayakawa, K. and Herzenberg, L. A. (1986). “Production of immunoglobulin isotypes by Ly-1+ B cells in viable motheaten and normal mice.” Science 232(4756): 1423-5.
Almost all B cells in autoimmune mice with the viable motheaten (mev) mutation express the Ly-1 cell surface antigen, which marks a minor population of B cells constituting a separate lineage in normal mice. Immunoglobulins primarily of the M and G3 classes, which in both normal and mev mice contain high levels of lambda light chain, are produced in excess in mev mice. These and other observations suggest that the development of B cells that express Ly-1 is regulated independently from the development of B cells that do not express Ly-1. B cells bearing the Ly-1 surface antigen may play specialized roles in the normal immune system and in autoimmunity by regulating other B cells via lymphokines, by producing antibodies to self and certain foreign antigens, and by preferentially secreting immunoglobulin M and immunoglobulin G3.
LAH #279-01
Bowcock, A. M., Kavathas, P., Margolskee, R. F., Herzenberg, L. and Cavalli-Sforza, L. L. (1986). “An RFLP associated with pcDLeu2-14, a human T-cell differentiation antigen CD8 (Leu2) cDNA mapped to 2p12.” Nucleic Acids Research 14(19): 7817.
Herzenberg, L. A., Kipps, T. J., Peterson, L. and Parks, D. R. (1985). Hybridoma variants affecting isotype, antigen binding and idiotype. Biotechnology in Diagnostics (Proceedings of the International Symposium on the Impact of Biotechnology on Diagnostics). H. Koprowski, S. Ferrone and A. Albertini. Amsterdam, Elsevier Science Publications. One: 3-16.
Nakauchi, H., Nolan, G. P., Hsu, C., Huang, H. S., Kavathas, P. and Herzenberg, L. A. (1985). “Molecular cloning of Lyt-2, a membrane glycoprotein marking a subset of mouse T lymphocytes: molecular homology to its human counterpart, Leu-2/T8, and to immunoglobulin variable regions.” Proceedings of the National Academy of Sciences of the United States of America 82(15): 5126-30.
The sequence of Lyt-2 cDNA shows that it is a new member of the immunoglobulin super gene family. Analysis of the predicted amino acid sequence indicates that the Lyt-2 polypeptide is synthesized with a 27-amino acid leader, and that the mature protein has an immunoglobulin variable region (Ig V)-related sequence of approximately 100 amino acids, an extracellular spacer of 43, a transmembrane region of 38, and an intracytoplasmic region of 27 amino acids. Lyt-2 and its human analogue Leu-2 are 56% homologous; analysis indicates that the Ig V-related domains of the two molecules have evolved away from each other faster than the carboxyl-terminal half of the proteins.
Bruns, G., Kavathas, P., Shiloh, Y., Sakai, K., Schwaber, J., Latt, S. A. and Herzenberg, L. A. (1985). “The human T cell antigen Leu-2 (T8) is encoded on chromosome 2.” Human Genetics 70(4): 311-4.
The locus encoding the human T lymphocyte cell surface antigen Leu-2 has been assigned to chromosome 2 with a DNA mapping panel derived from somatic cell hybrids. The two genomic components identified by a cDNA clone for Leu-2 segregated with human chromosome 2 in all 24 independent hybrid clones examined. The cosegregation of the Leu-2 and immunoglobulin kappa (IgK) loci in hybrids with spontaneous rearrangements of chromosome 2 is consistent with the possibility that the Leu-2 locus is on proximal human 2p near IgK. In the mouse, a locus for a T lymphocyte cell surface antigen with properties similar to Leu-2 is closely linked to the IgK locus on mouse chromosome 6. Hence the syntenic relationship of a gene implicated in T cell killing with the immunoglobulin kappa locus would then be conserved in the mouse and human genomes.
Herzenberg, L. A., Stall, A. and Lalor, P. (1987). Ly-1 B cells and autoimmunity. New Horizons in Animal Models for Autoimmune Disease, Academic Press: 149-157.
Hayakawa, K., Hardy, R. R. and Herzenberg, L. A. (1986). “Peritoneal Ly-1 B cells: genetic control, autoantibody production, increased lambda light chain expression.” European Journal of Immunology 16(4): 450-6.
Previous studies demonstrate that Ly-1 B cells and their progenitors are clearly detectable in peritoneum in normal mice. In this publication, we show (a) that peritoneal Ly-1 B cells resemble splenic Ly-1 B cells with respect to surface marker expression and functional activity (autoantibody production); (b) that Ly-1 B frequencies in peritoneum are considerably higher than in spleen; and (c) that genetic mechanisms reduce peritoneal Ly-1 B frequencies to minimal levels in SJL-related mice and to below detectability in CBA/N and other mice with the X-linked immunodeficiency (Xid). In addition, we show that that peritoneal (and perhaps splenic) Ly-1 B populations demonstrate an unique bias in immunoglobulin commitment. That is, they are selectively enriched for cells that express IgM heavy chains in association with lambda light chains. Thus, as a whole, evidence presented here defines the peritoneum as a tightly regulated lymphocyte compartment that normally houses a large population of mature Ly-1 B cells with distinctive functional properties.
Tagawa, M., Nakauchi, H., Herzenberg, L. A. and Nolan, G. P. (1986). “Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation.” Proceedings of the National Academy of Sciences of the United States of America 83(10): 3422-6.
We recently isolated the gene and a cDNA clone for the mouse T-cell surface antigen Lyt-2 and showed that Lyt-2 is homologous to the human Leu-2 (T8) antigen and that the gene encoding it is a member of the immunoglobulin gene superfamily. By screening a mouse thymus cDNA library with the Lyt-2 cDNA clone, we isolated two classes of cDNA clones, alpha and alpha', which differ by 31 base pairs. Comparison of the alpha cDNA with genomic sequence data indicates that there are five exons encoding Lyt-2: a fused leader/immunoglobulin variable region-like exon, a spacer region exon, a transmembrane exon, and two cytoplasmic exons. The alpha' cDNA clones lack the first of the two cytoplasmic exons and have a direct splice from the donor splice site of the transmembrane exon to the acceptor of the second cytoplasmic exon. This splice changes the reading frame for the second cytoplasmic exon, causing a stop codon shortly after the splice so that the alpha' cDNA clone codes for a peptide 25 residues shorter than the alpha cDNA-encoded peptide. We have constructed expression vectors with alpha and alpha' cDNAs and have shown that L-cell transfectants of these produce Lyt-2 polypeptides of the predicted sizes and that these associate as homodimers on the cell membranes. We found the two species of mRNA corresponding to alpha and alpha' cDNAs at equal levels in thymus RNA by using S1 nuclease analysis. Although lymph node T cells have only the alpha form of Lyt-2 protein, S1 nuclease analysis shows that lymph nodes have about 20% alpha' mRNA relative to alpha. Thus, Lyt-2 is regulated at RNA processing, translational, and/or post-translational steps.
Stall, A. M., Lalor, P. A., Herzenberg, L. A. and Herzenberg, L. A. (1986). “Ly-1 B cells and autoantibodies.” Annales d'Immunologie de l'Institut Pasteur 137D: 173-175.
Herzenberg, L. A., Stall, A. M., Lalor, P. A., Sidman, C., Moore, W. A. and Parks, D. R. (1986). “The Ly-1 B cell lineage.” Immunological Reviews 93: 81-102.