Herzenberg Publications 100-199close window to return to previous page |
LAH #100
Herzenberg, L. A., Okumura, K. and Herzenberg, L. A. (1975). Mechanism of Allotype Suppression. Symposium on Suppressor Cells in Immunity, London, Ontario, Canada, University of Western Ontario.
Stout, R. D. and Herzenberg, L. A. (1975). “The Fc receptor on thymus-derived lymphocytes. I. Detection of a subpopulation of murine T lymphocytes bearing the Fc receptor.” Journal of Experimental Medicine 142(3): 611-21.
Utilizing the fluorescence-activated cell sorter (FACS) and washed murine antibody-antigen complexes formed in antibody excess, we have demonstrated the presence of the Fc receptor on the surface of a distinct subpopulation of murine T lymphocytes. No differences in intensity of labeling with the complexes was observed when the Fc+ T lymphocytes were compared with Fc+ B lymphocytes. The majority of Fc+ T lymphocytes are small lymphocytes determined by light-scattering characteristics on the FACS. Separating Fc+ from Fc- T lymphocytes from spleens of mice primed 1 wk or 1 mo previously with keyhole limpet hemocyanin (KLH) revealed that the T cells capable of cooperating with DNP-KLH primed B cells to give an adoptive anti-DNP PFC response do not bear the Fc receptor.
Julius, M. H., Janeway, C. A., Jr. and Herzenberg, L. A. (1976). “Isolation of antigen-binding cells from unprimed mice. II. Evidence for monospecificity of antigen-binding cells.” European Journal of Immunology 6(4): 288-92.
Spleen cells from unimmunized mice were exposed to two contrastingly fluorescent antigens simultaneously. Antigen-binding cells of either specificity were isolated using a fluorescence-activated cell sorter (FACS). Purified cells binding one or the other of the antigens were then examined by fluorescence microscopy for the presence of bound antigen of the alternate specificity. No double binding cells were seen. If cells bear receptors of two or more specificities and these receptors are randomly distributed among antigen-binding cells, then of the 13 000 binding cells examined 82 were expected to bind both antigens. These results strongly suggest that antigen-binding cells bear receptors of only one specificity. In addition, by inference from the functional correlation between antigen-binding cells and precursor cells, the data support the contention that precursors of antibody-forming cells are monospecific.
Stout, R. D. and Herzenberg, L. A. (1975). “The Fc receptor on thymus-derived lymphocytes: II. Mitogen responsiveness of T lymphocytes bearing the Fc receptor.” Journal of Experimental Medicine 142(5): 1041-51.
The responsiveness of purified Fc- and Fc+ T lymphocytes, isolated from normal spleen cell populations by cell sorting on the fluorescence activated cell sorter, has been examined. Although both Fc- and Fc+ T cells responded to phytohemagglutinin, the response to concanavalin A (Con A) was found to be a characteristic of the Fc+ T lymphocyte. The poor responsiveness of the Fc- T cells to Con A was shown not to be due to a requirement of either different concentrations of Con A or for adherent cells. The addition of Fc+ T cells to the Fc- T cells in a ratio of 1:3 resulted in a mitotic response not significantly different from that observed with the purified Fc+ T cells alone and up to 15-fold greater than that of Fc- T cells alone. It is suggested that the Fc T cells can be recruited into mitosis as a result of Con A stimulation of the Fc+ T cells.
Loken, M. R., Sweet, R. G. and Herzenberg, L. A. (1976). “Cell discrimination by multiangle light scattering.” Journal of Histochemistry & Cytochemistry 24(1): 284-91.
Measurement of the light scattered by biological cells as a function of scattering angle provides information that can be correlated with cell type. Two flow systems that provide multiangle scattering data from cells have been constructed and tested. The first utilizes two narrow-aperture detectors positioned at different angles; the second utilizes the motion of the cell to generate complete scatter patterns of individual cells over a 67 degrees range of scattering angle.
Stout, R. D., Waksal, S. D. and Herzenberg, L. A. (1976). “The Fc receptor on thymus-derived lymphocytes. III. Mixed lymphocyte reactivity and cell-mediated lympholytic activity of Fc- and Fc+ T lymphocytes.” Journal of Experimental Medicine 144(1): 54-68.
The involvement of Fc- and Fc+ T cells, separated on the fluorescence-activated cell sorter, in proliferative and cytotoxic responses to alloantigens was examined. The cytotoxic lymphocytes generated by in vivo exposure to allogeneic tumor cells were shown to express the Fc receptor. The proliferative responses to alloantigen exposure in mixed lymphocyte cultures was equivalent in intensity for unseparated T cells, the Fc+ T-cell fraction, and the Fc- T-cell fraction isolated from nonsensitized spleen cells. In contrast, the cytotoxic responses generated by the Fc- T-cell fraction (less than 1% Fc+) were much weaker than the cytotoxic responses generated by the Fc+ T-cell fraction (80-90% Fc+), and the responses of the Fc+ T-cell fraction were generally weaker than, or equal to the responses of unseparated T cells (Fc- T less than Fc+ T less than or equal to unseparated T). Mixtures of the Fc- and Fc+ T-cell fractions mounted stronger cytotoxic responses than the sum of the responses of either fraction alone. Examination of the Ly phenotypes of the synergizing populations revealed that the CL precursor activity (Ly-2+ T cells) resided in the Fc- T-cell population, and that the amplifier T-cell activity (Ly-1+ T cells) resided in the Fc+ T-cell population. The data are discussed in terms of T-cell heterogeneity, differentiation, and intercellular interaction.
Sato, V. L., Waksal, S. D. and Herzenberg, L. A. (1976). “Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells.” Cellular Immunology 24(1): 173-85.
Petranyi, G. G., Keissling, R., Povey, S., Klein, G., Herzenberg, L. A. and Wigsell, H. (1976). “The genetic control of natural killer cell activity and its association with in viro resistance against a moloney lymphoma isograft.” Immunogenetics 3: 15-28.
Yutoku, M., Grossberg, A. L., Stout, R., Herzenberg, L. A. and Pressman, D. (1976). “Further studies on Th-B, a cell surface antigenic determinant present on mouse B cells, plasma cells and immature thymocytes.” Cellular Immunology 23(1): 140-57.
Okumura, K., Julius, M. H., Tsu, T. and Herzenberg, L. A. (1976). “Demonstration that IgG memory is carried by IgG-bearing cells.” European Journal of Immunology 6(7): 467-72.
Memory B cells which give rise to IgG antibody-producing cells were generally assumed to be IgG-bearing cells. However, recent studies indicating that very few IgG-bearing cells exist in lymphoid tissue brought this assumption into question. In this study, we examined directly the question whether IgG-bearing cells contain functional precursors of IgG antibody-producing cells. Using the adoptive secondary immune response, we demonstrated that Ig-1 b-bearing cells, isolated with the fluorescence-activated cell sorter (FACS), are the functional precursors of Ig- 1 b-producing cells. Further, we have enriched IgG2 and IgG1 memory B cells using the FACS. The results show that IgG2-bearing cells are the functional precursors of the IgG2 antibody-producing cells. Likewise, the IgG1-bearing cells are the functional precursors of IgG1 antibody-producing cells. Thus, IgG memory cells have surface IgG which indicates the class and allotype commitment of the memory cell and its progeny antibody-forming cells.
Okumura, K., Herzenberg, L. A., Murphy, D. B. and McDevitt, H. O. (1976). “Selective expression of H-2 (i-region) loci controlling determinants on helper and suppressor T lymphocytes.” Journal of Experimental Medicine 144(3): 685-98.
Data presented here show that locidentify in the I-region of the H-2 gene complex are selectively expressed in different functional T-cell subpopulations. These loci are closely linked (or possibly identical) to loci that control immune responses. They control surface determinants which identify helper and suppressor T lymphocytes. Determinants described here on allotype suppressor T cells (Ts) are found on normal (nonsuppressed) lymphoid cells, but are not found on helper T cells (Th). These determinants are controlled by a locus mapping in the I region of the H-2 complex. In an accompanying publication we show that this locus (Ia-4) marks a new I subregion (I-J) and is expressed only on T cells. Thus Ia-4 determinants idenfity a T-cell subpopulation which includes Ts but not Th. Th also carry identifying surface determinants controlled by loci that map to the H-2 complex, probably within the I region. These determinants are not found on Ts. Data presented also establish that loci in the I region control determinants on Th, but do not conclusively demonstrate that these are the determinants that distinguish Th from Ts. The selective expression of H-2-controlled determinants on Ts and Th suggests that these determinants are directly involved in immunoregulation.
Herzenberg, L. A., Okumura, K., Cantor, H., Sato, V. L., Shen, F. W. and Boyse, E. A. (1976). “T-cell regulation of antibody responses: demonstration of allotype-specific helper T cells and their specific removal by suppressor T cells.” Journal of Experimental Medicine 144(2): 330-44.
Allotype suppressor T cells (Ts) generated in SJL X BALB/c mice specifically suppress production of antibodies marked with the Ig-1a allotype. The studies presented here show that allotypes Ts suppress by specifically removing helper T cell (Th) activity required to facilitate differentiation and expansion of B cells to Ig-1b antibody-forming cells. We show first that Ts and Th belong to different T-cell subclasses as defined by Ly surface antigens. Ts are Ly2+Lyl- and thus belong to the same subclass as cytotoxic precursor and effector cells; Th are Lyl+Ly2- cells and thus belong to the subclass containing cells which can exert helper functions and initiate delayed hypersensitivity reactions. Placing these cells in these two subclasses shows that Th are different from Ts and suggests that they play different roles in regulating antibody responses. The difference in these roles is defined by the evidence presented here showing that Ts attack Th and regulate the antibody response by specifically regulating the availability of Th activity. We show that in allotype suppressed mice, Ts which suppress Ig-1b antibody production have completely removed the Th activity of helping Ig-1b cells without impairing Th activity which helps other IgB B cells. These findings imply the existence of allotype-specific Th for Ig-1b cells (Ig-1b Th). We directly establish that Ig-1b cells require such help by showing that carrier-primed spleen cells from Iga/Iga congenic hybrids help Ig-1a B cells from hapten-primed Igb/Iga donors but do not help Ig-1b B cells from the same donor in the same adoptive recipient.
Murphy, D. B., Herzenberg, L. A., Okumura, K. and McDevitt, H. O. (1976). “A new I subregion (I-J) marked by a locus (Ia-4) controlling surface determinants on suppressor T lymphocytes.” Journal of Experimental Medicine 144(3): 699-712.
In an accompanying publication we show that a subpopulation of T lymphocytes, which includes allotype suppressor T cells, selectively expresses I-region determinants. In this report, we show that these determinants are controlled by a new locus, Ia-4. Unlike the classically defined Ia antigens, they are not found on B lymphocytes. Antibody against Ia-4 determinants cannot be detected by conventional dye exclusion cytoxicity assays, suggesting that they are present on a small subpopulation (less than 10%) of peripheral T lymphocytes. The Ia-4 locus marks a new I subregion, provisionally designated I-J. This chromosomal segment is defined by the crossover positions in strains B10.A(5R) (K-end boundary) and B10.HTT (D-end boundary), and maps between the I-B and I-C subregions.
Cremer, N. E., Herzenberg, L. A. and McDevitt, H. O. (1976). “Fifteenth midwinter conference of immunologists: lymphocyte differentiation.” Clinical Immunology & Immunopathology 6(3): 431-42.
Herzenberg, L. A., Black, S. J., Loken, M. R., Okumura, K., van der Loo, W., Osborne, B. A., Hewgill, D., Goding, J. W., Gutman, G. and Warner, N. L. (1977). “Surface markers and functional relationships of cells involved in murine B-lymphocyte differentiation.” Cold Spring Harbor Symposia on Quantitative Biology 41 Pt 1: 33-45.
Murphy, D. B., Okumura, K., Herzenberg, L. A. and McDevitt, H. O. (1977). “Selective expression of separate I-region loci in functionally different lymphocyte subpopulations.” Cold Spring Harbor Symposia on Quantitative Biology 41 Pt 2: 497-504.
Determinants controlled by separate loci mapping in the I region of the H-2 gene complex are selectively expressed on subpopulations of lymphocytes which play different roles in generating humoral responses. The Ia-4 locus, which marks a new I subregion, designated I-J, controls determinants found on allotype suppressor and normal (nonsuppressed) T lymphocytes. These determinants are not present on helper T lymphocytes or B lymphocytes. In contrast, the Ia-1 locus, which marks the I-A subregion, controls determinants present on B lymphocytes but not on suppressor or helper T lymphocytes. Another locus (subregion not known) controls determinants which distinguish helper T lymphocytes from suppressor T lymphocytes. Such selective expression suggests that the products of these loci may plan an integral role in lymphocyte interactions.
Stout, R. D., Murphy, D. B., McDevitt, H. O. and Herzenberg, L. A. (1977). “The Fc receptor on thymus-derived lymphocytes. IV. Inhibition of binding of antigen-antibody complexes to Fc receptor-positive T cells by anti-Ia sera.” Journal of Experimental Medicine 145(1): 187-203.
Treatment of splenic T lymphocytes with anti-Ia antiserum inhibits the binding of antigen-antibody (AgAb) complexes to the majority (less than 50%) of Fc receptor-positive (FcR+) T cells. A similar inhibition was observed with anti-H-2D and anti-H-2K sera but not with anti-Thy 1.2. Despite the presence of Ia determinants on peripheral T cells, as established by the inhibition of AgAb binding, Ia could not be detected on peripheral T cells by immunofluorescence assays. Data obtained with the AgAb-binding inhibition assay indicate that determinants controlled by loci mapping in the I-A and I-C, S, or G regions are present on the FcR+ T cells. Evidence is presented that subpopulations of T cells within the FcR+ T-cell population may be distinguishable on the basis of which I-region-controlled determinant is expressed. The data are discussed in terms of phenotypic and functional heterogeneity of T lymphocytes.
Schroder, J. P. and Herzenberg, L. A. (1980). Prenatal diagnosis by cell sorting using a fluorescence-activated cell sorter(FACS). Genetic Disorders of the Fetus: Diagnosis, Precention and Treatment. New York, Plenum Press: 541-555.
Warner, N. L., Goding, J. W., Gutman, G. A., Warr, G. W., Herzenberg, L. A., Osborne, B. A., van der Loo, W., Black, S. J. and Loken, M. R. (1977). “Allotypes of mouse IgM immunoglobulin.” Nature 265(5593): 447-9.
Loken, M. R., Stour, R. D. and Herzenberg, L. A. (1979). Lymphoid cell analysis and sorting. Flow Cytometry and Sorting. M. Melamed, Mendelshon. New York, John H Wiley and Sons: 505-527.
Loken, M. R., Parks, D. R. and Herzenberg, L. A. (1977). “Identification of cell asymmetry and orientation by light scattering.” Journal of Histochemistry & Cytochemistry 25(7): 790-5.
Light scattering from chicken red blood cells has been used as a model system to identify the asymmetry of cells. The histogram for forward angle light scattering for these cells is bimodal, the signal size being dependent on the cell orientation. A dual orthogonal scatter system is used to conclusively demonstrate this orientational variation in signal. A third scattering system, using a single incident beam with two orthogonal detectors, is used to further characterize the orientational variation of the scatter signal. In this third system it is shown that the signal in a detector set 90 degrees from the incident beam collects light reflected from the cell surface. The optical selection of cells in specific orientations using these systems may circumvent the need to physically orient cell in flow systems.
Herzenberg, L. A., V.T. Oi (1977). “A Rapid and Efficient Method for Preparing Purified Radio-labeled antibody: Use in a Solid-phase Radioimmune assay.” `.
Herzenberg, L. A., L.A. Herzenberg (1978). Mouse immunoglobulin allotypes: Description and specific methodology. Handbook of Experimental Immunology. D. Weir. Oxford, England, Blackwell Scientific Publications: 12.1-23.
Loken, M. R., Parks, D. R. and Herzenberg, L. A. (1977). “Two-color immunofluorescence using a fluorescence-activated cell sorter.” Journal of Histochemistry & Cytochemistry 25(7): 899-907.
A technique for the analysis of fluorescein and rhodamine in a flow system using a single wavelength of excitation is described. Both optics and electronics are used to discriminate the fluorescein and rhodamine signals. This technique has been used to study the relationship between immunoglobulin M and immunoglobulin D on mouse splenic lymphocytes.
Goldsby, R. A., Osborne, B. A., Simpson, E. and Herzenberg, L. A. (1977). “Hybrid cell lines with T-cell characteristics.” Nature 267(5613): 707-8.
Golsby, R. A., Osborne, B. A., Simpson, E. and Herzenberg, L. A. (1977). Somatic Cell Hybrids with Tcell Characteristics. The Immune System: Genetics and Regulation. New Yokr, Academic Press: 265-271.
Herzenberg, L. A., L.A. Herzenberg (1976). Analysis and separation using the fluorescence activated cell sorter (FACS). Handbook of Experimantal Immunology. Oxford, England: 22.1-21.
Black, S. J., van der Loo, W., Loken, M. R. and Herzenberg, L. A. (1978). “Expression of IgD by murine lymphocytes. Loss of surface IgD indicates maturation of memory B cells.” Journal of Experimental Medicine 147(4): 984-96.
B lymphocytes capable of generating primary IgM and IgG plaque-forming cells (PFC) responses to burro erythrocytes have surface IgD, as do primary IgM PFC. IgG memroy cells arising after one injection of antigen are divided into two groups, one of which expresses surface IgD while the other has no detectable membrane IgD. PFC generated from the IgG memory cells lacking surface IgD show a higher average avidity than those arising from IgD-positive IgG memory cells, indicating that mature IgG memory cells do not have surface IgD. After more than one injection of antigen, few, if any, IgG memory cells have surface IgD. IgG PFC arising in primary or secondary immune response lack membrane-bound IgD. These data provide the outlines for a B-cell maturation pathway in which IgD marks unprimed and early memory B cells and is lost in mature memory cells. Studies presented here were conducted by isolating IgD+ and IgD- cells with the fluorescence-activated cell sorter and functional testing of the isolated populations in adoptive transfer experiments.
Black, S. J., Tokushia, T. and Herzenberg, L. A. (1980). “Memory B cells at successive stages of differentiation: expression of surface IgD and capacity for self renewal.” European Journal of Immunology 10(11): 846-51.
In recent studies, we have characterized two memory B cell populations capable of giving rise to IgG antibody-producing cells in adoptive recipients. One population carries surface IgD gives rise to predominantly low-affinity antibody responses; the other lacks detectable surface IgD and gives rise to predominatly high-affinity responses. These memory populations often coexist in individual donors for long periods of time; however, in strongly stimulated donors, the IgD+ population is lost after several weeks, and nearly all detectable B cell memory is IgD- thereafter. In this publication, we show that the IgD+ and IgD- memory populations represent B cells at two successive stages of antigen-dependent differentiation. We used the fluorescence-activated cell sorter (FACS) in a double isolation and transfer protocol to show directly that FACS-isolated IgD+ memory cells transferred to adoptive recipients give rise both to IgG antibody-producing cells and to an expanded memory population that is predominantly IgD-. We also show that FACS-isolated IgD- memory populations from the original donor "self-renew" (i.e. give rise to more IgD- memory) in adoptive recipients and that these events require supplementation of the isolated memory cells with carrier-primed T cells and antigen. In discussing these findings, we integrate our data with previous evidence on the expression of surface IgG on memory B cells to create an updated view of surface Ig expression during memory development. We also consider these findings in the light of our recent suggestion that the loss of IgD receptors facilitates affinity maturation in the more mature (IgD-) memory population.
Black, S. J., Goding, J. W., Gutman, G. A., Herzenberg, L. A., Loken, B. A., Osborne, W. and Warner, N. L. (1978). “Immunoglobulin isoantigen (allotypes) in the mouse V. Characterization of IgM allotypes.” Characterization of IgM allotypes.Immunogenetics 7: 213-230.
Herzenberg, L. A., L.A. Herzenberg, C. Milstein (1978). Cell hybrids of myelomas with antibody forming cells and T-lymphomas with T cells. Handbook of Experimental Immunology, 3rd Edition. Weir. Oxford, England, Blackwell Scientific Publications: 25.1-7.
Nahm, M. H., Herzenberg, L. A., Little, K. and Little, J. R. (1977). “A new method of applying the Sips equation.” Journal of Immunology 119(1): 301-5.
The Sips equation is frequently used in immunochemistry to describe the relationship between antibody-binding site concentration (n0[Ab]), antigen concentration, intrinsic affinity constant (K), and the heterogeneity index (a) of the affinity constant. Usually n0[Ab] is determined before calculating the remaining parameters (K and a). A new method is proposed which does not require knowledge of n0[Ab] nor an extensive calculation to determine K and a. The method can also be used to determine the antibody-binding site concentration without purified antibody or ligand saturation of the binding sites. This method can be applied to any antibody which binds a monovalent ligand and which can be obtained at a concentration greater than 1/K. Since the Sips equation can be applied to any ordinary chemical reaction by setting a=1, the proposed method can be used generally to determine the affinity constant and the initial concentration of one of the reactants.
Stout, R. D., Murphy, D. B., McDevitt, H. and Herzenberg, L. A. (1978). Ia antigens on FcR positive T Lymphocytes. Ir Genes and Ia Antigens. McDevitt. New York: 195-200.
LAH #134
Milstein, C. and Herzenberg, L. A. (1977). T and B Cell Hybrids. Regulatory Genetics of the Immune System: ICN-UCLA Symposia on Molecular and Cellular Biology, Los Angeles, CA, Academic Press.
van der Loo, W., Gronowicz, E. S., Strober, S. and Herzenberg, L. A. (1979). “Cell differentiation in the presence of cytochalasin B: studies on the "switch" to IgG secretion after polyclonal B cell activation.” Journal of Immunology 122(4): 1203-8.
Mouse spleen cells were cultured with lipopolysaccharide in conditions that activate both IgM and IgG secretion. Addition of cytochalasin B (CB), an inhibitor of cytokinesis, lead to a high degree of polynucleation, with little effect on Ig secretion. Using cytoplasmic staining with fluorochrome conjugated antisera, we determined the numbers of IgG-containing cells that also contained IgM in their cytoplasm. Such double staining cells were relatively more frequent at early times of the cultures, but at all times single producing cells were in the majority. Addition of CB over the period when the IgG producing cells first appear, lead to a marked increased frequency of double staining, polynucleated cells. This characteristic was stable over a period of at least 42 hr, suggesting that each double staining cell actively synthesized both isotypes. When CB was added after IgG production had started, little increase in the numbers of double staining cells were observed, although polynucleation remained extensive. These data confirm previous findings that the lineage of one cell can produce both IgM and IgG. Furthermore, the results suggest that cells in the process of switching from IgM to IgG go through an asymmetric division leading to one IgM-producing and one IgG-producing daughter cell.
Herzenberg, L. A., L. Wofsy (1977). Cell separation and characterization. mImmune System: Genetics and Regulation. New York, Academic Press: 341-353.
Stovel, R. T., Sweet, R. G. and Herzenberg, L. A. (1978). “A means for orienting flat cells in flow systems.” Biophysical Journal 23(1): 1-5.
Flattened cells, such as red blood cells, epithelial cells, and sperm of many species, cause problems for fluorescence-activated cell analysis and sorting machines because the flow systems of such devices are unable to control the orientation of these cells as they flow past the detectors. For this reason, the fluorescence or scattered light measurements for identical cells may vary greatly. A flow geometry is here described that orients flat cells in a coaxial flow system so that each cell presents the same aspect to the observation device. A wedge-shaped exit on the sample injection tube in a coaxial flow system is sufficient to produce the desired orientation effect when used with low sample flow rates. Data is presented showing the effect of orientation of fixed chicken erythrocytes on histograms of small forward-angle light-scattering measurements.
Herzenberg, L. A., Bianchi, D. W., Schroder, J., Cann, H. M. and Iverson, G. M. (1979). “Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting.” Proceedings of the National Academy of Sciences of the United States of America 76(3): 1453-5.
Fetal cells, potentially usable for prenatal diagnosis, were sorted from maternal blood samples taken as early as 15 weeks of gestation. Immunogenetic and cytogenic criteria established the fetal origin of the observed cells: Y-chromatin-containing (male) cells were detected in the sorted sample if and only if the newborn proved to be male and carried cell-surface antigens detected by the fluorescent-labeled antibody used for sorting with the fluorescence-activated cell sorter.
Oi, V. T., Jones, P. P., Goding, J. W. and Herzenberg, L. A. (1978). “Properties of monoclonal antibodies to mouse Ig allotypes, H-2, and Ia antigens.” Current Topics in Microbiology & Immunology 81: 115-20.
Iverson, G. M., Goldsby, R. A. and Herzenberg, L. A. (1978). “Expression of Thy 1.2 antigen on hybrids of B cells and a T lymphoma.” Current Topics in Microbiology & Immunology 81: 192-4.
Osborne, B. A., Goldsby, R. A. and Herzenberg, L. A. (1978). “Selective expression of loci in th I--J region on T cell hybrids.” Current Topics in Microbiology & Immunology 81: 217-20.
Herzenberg, L. A. (1978). “The fluorescence-activated cell sorter (FACS): a retrospective and prospective view. pp. 99-109.” In: Knapp W, et al., ed. Immunofluorescence and related staining techniques. Amsterdam, Elsevier/North-Holland. http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=0000370288.
LAH #142-01
Tsu, T. and Herzenberg, L. A. (1979). Solid-Phase Radiioimmune Assays. Additional Methods: 373-397.
Goding, J. W., Oi, V. T., Jones, P. P. and Herzenberg, L. A. (1979). Monoclonal antibodies to alloantigens and to immunoglobulin allotypes. pp. 309-31. In: Pernis B, Vogel HJ, ed. Cells of immunoglobulin synthesis. New York, Academic Press. http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=0000381447.
Herzenberg, L. A. (1979). “Seek well and ye shall find [editorial].” New England Journal of Medicine 300(25): 1435-6.
Kontiainen, S., Simpson, E., Bohrer, E., Beverley, P. C., Herzenberg, L. A., Fitzpatrick, W. C., Vogt, P., Torano, A., McKenzie, I. F. and Feldmann, M. (1978). “T-cell lines producing antigen-specific suppressor factor.” Nature 274(5670): 477-80.
LAH #144
Oi, V., T. and Herzenberg, L. A. (1979). Hybrid Cell Lines. Selected Methods in Cellular Immunology. B. B. Mishell, W. H. Shiigi and W. H. Freeman. San Francsisco, W.H. Freeman Publishing: 17.1-17.11.
Goldsby, R. A., Osborne, B. A., Suri, D., Mandel, A., Williams, J., Gronowicz, E. and Herzenberg, L. A. (1978). “Production of specific antibody without specific immunization.” Current Topics in Microbiology & Immunology 81: 149-51.
Simpson, E., Kontiainen, S., Herzenberg, L. A., Bohrer, E., Torano, A., Vogt, P., Beverley, P., Fitzpatrick, W. and Feldmann, M. (1978). “T cell hybrids with T cell functions.” Current Topics in Microbiology & Immunology 81: 195-202.
Goding, J. W., Black, S. J. and Herzenberg, L. A. (1978). Biosynthesis and two-dimensional gel analysis of IgM and IgD receptors. B Lymphocytes in the Immun Response. New York, Elsevier North-Holland: 9-13.
Black, S. J. Takeshi Tokuhisu and Herzenberg, L. A. (1979). “Regulation of B Cell Differentiation.” B Lymphocytes in the Immune Response Eds: Cooper/Mosier/Scher/Vietta, Elvesierf, North Holland. 147-9.
Black, S. J. and Herzenberg, L. A. (1979). “B-cell influences on the induction of allotype suppressor T cells.” Journal of Experimental Medicine 150(1): 174-83.
Hance, A. J., Robin, E. D., Simon, L. M., Alexander, S., Herzenberg, L. A. and Theodore, J. (1980). “Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.” Journal of Clinical Investigation 66(6): 1258-64.
Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 2.7.1.40) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11 +/- 0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 +/- 0.04 U/microgram DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +/- 0.13; hypoxia, 0.94 +/- 0.13 microgram enzyme protein/microgram DNA). Specific activity was not significantly different after 96 h (L2: normoxia, 261 +/- 11; hypoxia, 261 +/- 14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic O2 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to charcterizing enzymes, as well as quantitating cellular enzyme content.
Herzenberg, L. A., A.P. Dowsett, S.J. Black, L.A. Herzenberg (1979). Genetic and serologic requirements for indiction of spontaneous and anti-Ig induced allotype suppression,. B Lymphocytes in the immune response. New York, Elsvier North-Holland: 315-322.
Parks, D. R., Bryan, V. M., Oi, V. T. and Herzenberg, L. A. (1979). “Antigen-specific identification and cloning of hybridomas with a fluorescence-activated cell sorter.” Proceedings of the National Academy of Sciences of the United States of America 76(4): 1962-6.
Myeloma-spleen cell hybrids (hybridomas) producing antibody to mouse immunoglobulin allotypes have been labeled with fluorescent microspheres coupled with myeloma protein antigens. The ratio of specific to nonspecific microsphere binding by viable hybridoma cells was about 100:1. By using a modified fluorescence-activated cell sorter (FACS), selected hybridoma cells in a mixture have been sorted individually into media in microculture wells, where, with thymocyte feeder cells, they developed into clones producing a desired monoclonal antibody. Viable cells were selected by measurement of their light scattering and autofluorescence properties. Rare antibody-producing clones were obtained without laborious screening and repeated subculturing. This technique should expand the range of monoclonal antibodies readily obtained from hybridomas and greatly facilitate the process of obtaining desired hybridomas.
Ledbetter, J., Goding JW, Tsu TT, Herzenberg LA (1979). “A new mouselymphoid alloantigen (Lgp100) recognized by a monoclonal rat antibody.” Immunogenetics 8: 347-360.
Herzenberg, L. A. and Black, S. J. (1980). “Regulatory circuits and antibody responses.” European Journal of Immunology 10(1): 1-11.
Herzenberg, L. A., S.J. Black, T. Tokuhisa, L.A. Herzenberg (1979). Regulatory circuits and affinity maturation. Proceedings Columbia University College of Physicians and Surgeons Biomedical Symp., June 8-10, 1979. Regulatory T Lymphocytes. P. a. Vogel. New York, Academic Press: 417-439.
Oi, V. T. and Herzenberg, L. A. (1979). “Localization of murine Ig-1b and Ig-1a (IgG 2a) allotypic determinants detected with monoclonal antibodies.” Molecular Immunology 16(12): 1005-17.
Ledbetter, J. A. and Herzenberg, L. A. (1979). “Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens.” Immunological Reviews 47: 63-90.
Herzenberg, L. A., J.A. Ledbetter (1979). Monoclonal antibodies and the fluorescence-activated cell sorter: Complementary tools in lymphoid cell biology. Molecular Basis of Immune Cell Function. Amsterdam, Elsevier/North-Holland Biomedical Press: 315-330.
Ledbetter, J., Goding JW, Tokuhisa T, Herzenberg LA (1980). Murine T cell differentiation and antigens detected by monoclonal antibodies. Monoclonal Antibodies. New York, Plenum Publishing Corp: 235-249.
Herzenberg, L. A., S.J. Black, L.A. Herzenberg (1980). Regulation of antibody responses. Strategies of Immune Regulation. New York: 157-177.
LAH #158a
Herzenberg, L. A., S.J. Black, T. Tokuhisa, L.A. Herzenberg (1980). A role of IgD: Dissociation of memory induction from affinity maturation. Strategies of Immune Regulation. New Yokr, Academic Press: 439-441.
Micklem, H., S., Ledbetter, J. A., Eckhardt, L. A. and Herzenberg, L. A. (1980). Analysis of lymphocyte subpopulations with monoclonal antibodies to thy-1, lyt-1 and ThB antigens. Regulatory T Lymphocytes. New York, Academic Press: 119-132.
Tokuhisa T, O. V., Gadus FT, Herzenberg LA. (1980). Induction of allotype suppression with monoclonal antibodies. Regulatory T Lymphocytes. New York, Academic Press: 315-328.
Herzenberg, L. A., Black, S. J. and Tokuhisa, T. (1980). “Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors.” Journal of Experimental Medicine 151(5): 1071-87.
Goding, J. W. and Herzenberg, L. A. (1980). “Biosynthesis of lymphocyte surface IgD in the mouse.” Journal of Immunology 124(6): 2540-7.
The synthesis of IgD was studied in mouse spleen cells by using [35S]methionine labeling followed by immunoprecipitation with monoclonal antibody and two-dimensional gel electrophoresis. After a 15-min pulse of [35S]methionine, a relatively basic form (IgD1) of apparent m.w. 59,000 was precipitated. Conversion into more acidic forms of m.w. 63 to 72,000 (IgD2) took place during a chase period of several hours. The acidic form was identical in mobility to that of IgD labeled by surface radioiodination, and was almost completely removed by treatment of intact cells with pronase. Neuraminidase treatment of the surface form (IgD2) produced a form resembling IgD1 in charge, but with no detectable change in m.w. Treatment of IgD1 with endoglycosidase H resulted in a form with an apparent m.w. of 50,000, whereas IgD2 was resistant to this enzyme. Both IgD1 and IgD2 bound to lentil lectin, whereas only IgD2 bound to Ricinus communis hemagglutinin, which binds to terminal galactose residues. These results indicate that IgD is synthesized as an incompletely glycosylated precursor possessing "high mannose" type oligosaccharide moieties, and passes relatively slowly through the cell. Shortly before surface appearance, galactose and sialic acid are added. No specific association with any other labeled protein was observed, and any IgD secretion was below the limits of detectability.
Herzenberg, L. A. and Tokuhisa, T. (1980). “Carrier-priming leads to hapten-specific suppression.” Nature 285(5767): 664-7.
LAH #165
Herzenberg, L. A., S.J. Black , L.A. Herzenberg (1979). Circuits for regulation antibody responses. Molecular Basis of Immune Cell Function, Elseveir/North-Holland Biomedicaln Press: 147-160.
Oi, V. T., Bryan, V. M. and Herzenberg, L. A. (1980). “Lymphocyte membrane IgG and secreted IgG are structurally and allotypically distinct.” Journal of Experimental Medicine 151(5): 1260-74.
We have demonstrated that there are structurally distinct membrane and secreted IgG2a immunoglobulin molecules. The membrane heavy chain is both larger and more acidic than the secreted molecule. This difference is not a result of different N-glycosidic-linked oligosaccharide chains. The membrane heavy chain also is antigenically different from its secreted homologue. This is based on the fact that secreted IgG2a molecules express an allotypic determinant absent on membrane molecules. We discussed the genetic control and gene organization of membrane and secreted immunoglobulin heavy chain sequences and suggest mechanisms controlling the expression of the simian virus 40 genome as models for alternate gene expression of membrane and secreted heavy chain polypeptide chains from the same DNA sequence. The possible biological significance of the membrane immunoglobulin acting as a recognition site for regulatory T cells also is discussed. The difference between membrane and secreted immunoglobulin is proposed as a possible explanation for the manner in which T cells interact with IgG on memory B cells in the presence of a large excess of IgG present in body fluids.
Ledbetter, J. A., Evans, R. L., Lipinski, M., Cunningham-Rundles, C., Good, R. A. and Herzenberg, L. A. (1981). “Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man.” Journal of Experimental Medicine 153(2): 310-23.
Eckhardt, L. A. and Herzenberg, L. A. (1980). “Monoclonal antibodies to ThB detect close linkage of Ly-6 and a gene regulating ThB expression.” Immunogenetics 11(3): 275-91.
We have generated three hybridomas producing rat monoclonal antibodies to a surface antigen, ThB, that is shared by murine B lymphocytes and approximately 50 percent of murine thymocytes. These antibodies, produced by immunizations with MOPC-104E cells, appear to recognize the same antigen that was previously detected by rabbit and goat antisera to MOPC-104E cells (Yutoku et al. 1974, Yutoku et al. 1976). Using these antibodies, we have studied a genetic polymorphism that is associated with the level of ThB expression on B lymphocytes but not with the antigen's expression on thymocytes. We present evidence that this trait is controlled by one gene, Thb, which we find to be very closely linked to the gene or genes controlling the Ly-6, Ly-8, DAG, and Ala 1 antigen(s). While the latter four antigens were described as markers on mature T (or activated T and B) lymphocytes, ThB is restricted to immature thymocytes and all B cells. ThB is not expressed on kidney, although some investigators (McKenzie et al. 1977 a, Halloran et al. 1978) report Ly-6 expression on that tissue. SJL/J, C57BL/10JHz, DBA/2J, and AKR/J are among the mouse strains carrying the Thbh allele, while BALB/cN, CBA/J, C3H.SW/SnHz, and A/J carry the Thb1 allele. The ThB antigen has not yet been identified as a glycoprotein after cell-surface iodination, NP-40 solubilization, and immunoprecipitation.
Howard, F. D., Ledbetter, J. A., Mehdi, S. Q. and Herzenberg, L. A. (1980). “A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes.” Journal of Immunological Methods 38(1-2): 75-84.
Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favorably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants.
Ledbetter, J. A., Rouse, R. V., Micklem, H. S. and Herzenberg, L. A. (1980). “T cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens. Two-parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views.” Journal of Experimental Medicine 152(2): 280-95.
Using monoclonal antibodies and multiparameter fluorescence analyses, we show that the expression of Lyt-1, Lyt-2, and Lyt-3 on T cell subpopulations is more complex than was originally recognized by the cytotoxic depletion studies with conventional reagents that defined the Lyt-1+2+3+, Lyt-1+2-3-, and Lyt-1-2+3+ populations. We detect at least some Lyt-1 on all T (Thy-1-bearing) lymphocytes; however, in agreement with previous studies, we find that Lyt-2+3+ cells are more difficult to depelete with anti-Lyt-1 than Lyt-1+2-3- cells. Surprisingly, we found a small subpopulation of cells carrying relatively large amounts of Lyt-1 and no Thy-1 detectable by fluorescence-activated cell sorter analysis. We also detect cells with this phenotype histologically in B cell zones (primary follicles) and germinal centers in spleen and lymph nodes. In general, the Lyt-1 only population represents approximately 2% of spleen cells. The relative quantitative expression of Thy-1, Lyt-1, Lyt-2, and Lyt-3 changes systematically during T cell maturation. Among Lyt-1+2+3+ cells in the thymus, Thy-1 and Lyt-2 are high, whereas Lyt-1 is low. Among splenic T cells, in contrast, Thy-1 is low, Lyt-1 is high, and Lyt-2 and Lyt-3 are a little higher than in thymus. In general, Thy-1 expression is negatively correlated with Lyt-1. Thus, even among splenic and lymph node T cells subpopulations exist that tend to be either high Thy-1 and low Lyt-1 or vice versa. Lyt-2+3+ cells represent approximately 85% of thymocytes but only approximately 35% of splenic or lymph node T cells. The Lyt-2+3+ cells are found predominantly in the low Lyt-1, high Thy-1 subpopulation.
LAH #171
Habu, S., Kasai, M., Nagai, Y., Tamaoki, N., T, T. A., Herzenberg, L. A. and Okumura, K. (1980). “The glycolipid asialo GM1 as a new differentiation antigen of fetal thymocytes.” Journal of Immunology 125(5): 2284-8.
Antibody directed to the neutral glycolipid "asialo GM1" was found to react with the majority of thymus lymphoid cells lacking characteristic T cell markers in mice at an early embryonic stage (13 days of gestation). The proportion of these asialo GM1-positive cells (asialo GM1+ cells) decreased strikingly thereafter, contrasting with an increased fraction of lymphocytes possessing T cell surface markers such as Thy-1, Lyt-1, and Lyt-2. After about 18 gestational days, only a few asialo GM1+ cells were detected in the thymus as well as in other lymphoid organs. Double-staining analysis of the embryonic thymocytes (13 days of gestation) with anti-asialo GM1 and anti-Thy-1 demonstrated that thymocytes stained with anti-asialo GM1 did not react with anti-Thy-1, and vice versa. Morphologic examination by immunoelectronmicroscopy demonstrated that these asialo GM1+ cells were mainly composed of immature, large lymphoid cells having large nucleoli and relatively abundant cytoplasm with many polysomes. These results suggest that asialo GM1 is present on very early thymocytes and is lost as the mature murine T cell protein antigens Thy-1, Lyt-1, and Lyt-2 develop on these cells. The antibody to this glycolipid is a useful tool for studying embryonic thymic differentiation.
Parsons, M. and Herzenberg, L. A. (1981). “A monoclonal mouse antiallotype antibody reacts with certain human and other vertebrate immunoglobulins: genetic and phylogenetic findings.” Immunogenetics 12(3-4): 207-19.
A new mouse monoclonal antibody, 18.1, recognizes an allotypic determinant on mouse IgG1 of the "a" allotype. We found that this alloantibody reacts with immunoglobulins of evolutionarily distant vertebrate species including man, and with only certain isotypes or allotypes in some of these species. Most mammalian sera are reactive, except those from lagomorphs, marsupials, and monostremes. Avian and reptilian sera are also positive, while the amphibian and fish sera tested were negative. In human sera, 18.1 detects the isotypic marker on IgG2 and an allotypic marker on IgG3. This reactivity parallels the distribution of the Gm non-g determinant. These findings are discussed in relation to the phylogeny of IgG and its subclasses.
Herzenberg, L. A., J.A. Ledbetter (1981). Homologous T cell differentiation antigens of mouse and man. Frontiers in Immunology. W. E. Hildemann. New York, Elsevier/North-Holland: 179-188.
Howard, F. D., Ledbetter, J. A., Wong, J., Bieber, C. P., Stinson, E. B. and Herzenberg, L. A. (1981). “A human T lymphocyte differentiation marker defined by monoclonal antibodies that block E-rosette formation.” Journal of Immunology 126(6): 2117-22.
A new human T cell surface antigen, Leu-5, has been defined using a set of monoclonal antibodies that block rosette formation between T lymphocytes and sheep erythrocytes (SRBC). Four antibodies obtained from 2 different fusions using 2 immunized mouse strains all reacted with the same antigen. All these antibodies gave identical quantitative immunofluorescence (FACS) profiles, all gave the same staining profiles and intensities when used singly or in combinations, and all precipitated the same molecule. The antigen is a single polypeptide chain, 40,000 to 50,000 Mr, and is found on all thymocytes, peripheral T cells, and some null cells, but not on B cells. Leu-5 is a differentiation antigen that decreases in density as thymocytes mature to peripheral T lymphocytes. Thus, the Leu-5+ subpopulations ranked in order of decreasing Leu-5 density are: a subpopulation of subcapsular thymocytes greater than cortical and medullary thymocytes greater than peripheral T cells (cytotoxic/suppressor subset) greater than peripheral T cells (helper/inducer subset). The density distribution pattern of Leu-5 parallels the relative affinity of thymocytes and peripheral T lymphocytes for SRBC. We suggest that Leu-5 is either identical to or closely associated with the human T lymphocyte receptor for SRBC.
Iverson, G. M., Bianchi, D. W., Cann, H. M. and Herzenberg, L. A. (1981). “Detection and isolation of fetal cells from maternal blood using the flourescence-activated cell sorter (FACS).” Prenatal Diagnosis 1(1): 61-73.
The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.
Haaijman, J. J., Micklem, H. S., Ledbetter, J. A., Dangl, J. L. and Herzenberg, L. A. (1981). “T cell ontogeny. Organ location of maturing populations as defined by surface antigen markers is similar in neonates and adults.” Journal of Experimental Medicine 153(3): 605-14.
Earlier studies have suggested that splenic T cell populations in nursling mice (less than 18 d of age) have Lyt cell surface antigens that identify them as less mature than their adult counterparts. Studies presented here, however, demonstrate that the expression of the Thy-1, Lyt-1, Lyt-2, and Lyt-3 T cell antigens is virtually identical in 14-d-old and adult T cell populations even though at 14 d, T cells constitute less than 10% of the total spleen cell population. Because the expression of these antigens on the immature (cortical) thymocyte population differs substantially from their expression on peripheral T cells, the maturity of splenic T cells as judged by these criteria is similar in nurslings and adults. Very few cells in the neonatal thymus 4 h after birth correspond, in terms of antigen expression, to the more mature (medullary) thymocyte population of adults, but such cells develop rapidly during the first few days of life. They are present, therefore, sufficiently early to serve as the immediate source of peripheral T cells, as they apparently do in the adult. This then suggests that the locations for the major T cell maturational events are established within the first 2 wk of life of the mouse and maintained as such thereafter. The use of monoclonal antibodies and quantitative immunofluorescence analysis in our studies probably explains the differences between our findings and those reported previously, which relied on cytotoxic depletion by alloantisera and complement to estimate the frequencies of cells carrying the Lyt differentiation antigens in nurslings.
Lipinski, M. and Herzenberg, L. A. (1981). “Les hybridomes et leurs applications.” La Recherche 125: 952-961.
Herzenberg, L. A., T. Tokuhisa, F.T. Gadus, S.J. Black, L.A. Herzenberg (1981). Regulation of memory B cell development and expression. Frontiers in Immunology. W. H. a. F. Hildemann, J.A. New York, Elsevier/North Holland, Inc.: 75-90.
Lanier, L. L., Warner, N. L., Ledbetter, J. A. and Herzenberg, L. A. (1981). “Expression of Lyt-1 antigen on certain murine B cell lymphomas.” Journal of Experimental Medicine 153(4): 998-1003.
Although the Lyt-1 antigen has previously been considered a T cell-specific marker, recent evidence suggests that a population of Thy-1-, Lyt-1+ cells exists in normal lymphoid tissues. In this study, we have observed that the WEHI-55, WEHI-259, and CH5 B cell lymphomas express high levels of the Lyt-1 antigen, as detected by monoclonal antibodies using the fluorescence activated cell sorter. Three other B cell lymphomas of the 11 examined also gave weak but detectable reactions with the anti Lyt-1 monoclonal antibody. Except for the expression of the Lyt-1 antigen, these lymphomas are typical of cells in the B cell lineage with respect to surface phenotype. The Lyt-1 glycoprotein immunoprecipitated from metabolically labeled WEHI-55 cells is similar in structure to the Lyt-1 glycoprotein on thymocytes. These findings are similar to recent reports that B-type human lymphocytic leukemia cells express the putative human homologue of Lyt-1, the Leu-1 antigen.
Herzenberg, L. A., T. Takeshi, D.R. Parks, L.A. Herzenberg (1981). Hapten-specific regulation of heterogeneous antibody responses: Intersection of Theory and Practices. The Immune System. Basel, Switzerland, S. AG.
Ledbetter, J. A., Seaman, W. E., Tsu, T. T. and Herzenberg, L. A. (1981). “Lyt-2 and lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds. Relationship of subunits to T cell cytolytic activity.” Journal of Experimental Medicine 153(6): 1503-16.
Lyt-2 and Lyt-3 antigens are carried on separate disulfide-bonded subunits of the same cell surface macromolecules. These are present on thymocytes in a variety of multimeric forms consisting of disulfide-bonded dimers, tetramers, and hexamers of pairwise combinations of three subunits (30,000, 34,000, and 38,000 Mr). From reduced and alkylated Nonidet-P40 thymus extracts, a monoclonal anti-Lyt-3 precipitates only the 30,000 Mr subunit, but not the 30,000 Mr subunit. Almost all of the Lyt-2 and Lyt-3 subunits on the cell are covalently linked by disulfide bonds. However, small amounts of free Lyt-3 subunit was seen in some experiments. Similarly, small amounts of Lyt-2-3- material, consisting of dimers of the 38,000 and 34,000 Mr subunits were identified. Each of the three subunits migrated with a basic charge (pI greater than 8) on two-dimensional gels. Cytotoxic effector cells that are blocked by anti-Lyt-2 and anti-3 can be treated with trypsin and other arginine-specific proteases to remove these antigens. At low concentrations of these proteases, Lyt-3 antigens are selectively removed. After selective removal of Lyt-3 antigens, cytotoxic effector cells are still active and blocking of activity by anti-Lyt-3 is significantly reduced, whereas blocking of activity by anti-Lyt-2 is significantly increased. Neither Lyt-2 nor Lyt-3 is allelically excluded on thymocytes or T cells. These results suggested that the Lyt-2, Lyt-3 macromolecules are associated with but do not serve as the T cell antigen receptor.
Herzenberg, L. A., T. Tokuhisa, L.A. Herzenberg (1981). Carrier specific induction of hapten-specific suppression. T Lymphocytes Today. J. Inglis. New York, Elsevier Science: 77-83.
Dowsett, A. P. and Herzenberg, L. A. (1981). “Regulation of the production of murine IgG2a by an H-2-linked gene and other unlinked genes.” Immunogenetics 13(3): 237-45.
Alleles of at least two loci (rig-1 and Rig-2) regulate the levels of serum immunoglobulin of the Igh-1b class and allotype in BALB/c Igb (BAB/14) and (BALB/c x BAB/14)F1 mice. The combined effect of the BALB/c alleles at these two loci is to lower Igh-1b levels significantly below those observed in other strains and below their own levels of Igh-1a in allotype heterozygous mice. The rig-1 locus is closely linked to or within the H-2 complex. Two alleles have been defined: rig-1d and rig-1b in H-2d and H-2b haplotypes, respectively. Homozygous rig-1d animals heterozygous for the BALB/c Rig-2 allele(s) have very low levels of Igh-1b. The designation of Rig-2 is provisional since it has not been mapped or defined as a single locus.
Herzenberg, L. A., T. Tokuhisa, L.A. Herzenberg (1981). Hapten-specific regulation of memory B cell expression. B Lymphocytes in the Immune Response: Functional, Developmental and Interactive Properties. M. D. Klinman N, Scher I, Vittetta E. New York, Elsevier/North Holland: 411-413.
Seaman, W. E., Talal, N., Herzenberg, L. A. and Ledbetter, J. A. (1981). “Surface antigens on mouse natural killer cells: use of monoclonal antibodies to inhibit or to enrich cytotoxic activity.” Journal of Immunology 127(3): 982-6.
In studies using monoclonal antibodies to cell-surface antigens we have identified 2 new antigens (H11 and 7.2) expressed on mouse NK cells. These are shared with T cells but not B cells. We have also shown that NK cells express T200 but lack detectable ThB or Lyt-1. The T200 and H11 surface molecules were implicated in the mediation or regulation of natural killing; monoclonal antibodies to T200 and H11 inhibited natural killing when added to the cytotoxicity assay but did not interfere with T cell cytotoxicity against the same target. The inhibitory effect of anti-T200 is consistent with recent evidence showing that antibodies to the Ly-5 polymorphic determinant on T200 block natural killing. The H11 determinants is on a different molecule. The absence of Lyt-1 and ThB on NK cells permitted development of a rapid and simple method for separating NK cells from T cells and B cells. Spleen cells incubated with rat monoclonal antibodies to Lyt-1 (on all T cells) and ThB (on all B cells) were 95% removed by adherence to Petri dishes coated with antiserum to rat immunoglobulin. The natural killer activity in the nonadherent population was enriched 16-fold.
Lipinski, M., Parks, D. R., Rouse, R. V. and Herzenberg, L. A. (1981). “Human trophoblast cell-surface antigens defined by monoclonal antibodies.” Proceedings of the National Academy of Sciences of the United States of America 78(8): 5147-50.
A series of monoclonal antibodies has been raised against the human choriocarcinoma cell-line, BeWo. Four antigens, Trop-1, -2, -3, and -4, are defined on normal and malignant trophoblast cells. Trop-1 and Trop-2 appear to be specifically expressed on syncytio- and cytotrophoblasts, whereas Trop-3 and Trop-4 are also detected on various tumor cell lines, normal lymphocytes, and monocytes. Anti-Trop-1 and anti-Trop-2 antibodies might prove useful for detection and isolation of fetal trophoblast cells circulating in pregnant women's blood and for diagnosis and therapy in patients having choriocarcinomas and other germ-cell neoplasms.
Ledbetter, J., Frankel AE, Herzenberg LA, Herzenberg LA (1981). Human Leu T cell differentation antigens - Quantitative expression in normal lympoid cells and cell lines. Monoclonal Antibodies and T Cell Hybridomas. H. U. Hammerling GJ, Kearney K. New Yoek, Elsevier/North Holland: 16-22.
Parks, D. R. and Herzenberg, L. A. (1982). “Fetal cells from maternal blood: their selection and prospects for use in prenatal diagnosis.” Methods in Cell Biology 26: 277-95.
Herzenberg, L. A., C.M. Huang, V.T. Oi, M. Parsons (1981). The structure and genetics of mouse immunogobulin heavy chain constant regions defined by monoclonal anti-allotype antibodies. Immunoglobulin Idiotypes and Their Expression: ICN-UCLA Symposia on Molecular and Cellular Biology. S. E. Janeway C, Wigzell H., Fox CF,. New York, Academic Press. XX: 199-208.
Lanier, L. L., Warner, N. L., Ledbetter, J. A. and Herzenberg, L. A. (1981). “Quantitative immunofluorescent analysis of surface phenotypes of murine B cell lymphomas and plasmacytomas with monoclonal antibodies.” Journal of Immunology 127(4): 1691-7.
In this study, a large series of murine B lymphomas and plasmacytomas were examined by quantitative flow cytometry analysis using a panel of monoclonal antibodies against murine differentiation antigens. These cell lines appear to represent subpopulations of lymphoid cells arrested at distinct stages of differentiation. In general, the pattern of reactions of these monoclonal antibodies with the B cell neoplasms was comparable to the reactions seen with normal cells in the same lineage. The Thy-1.2, Lyt-2, and T-30 differentiation antigens were not detected on any B lymphoma or plasmacytoma. However, certain surface Ig-positive B lymphomas do express the Lyt-1 antigen. With respect to other differentiation markers examined, including E2, F1, ThB, Lgp-100 (Ly 9.1), G2, and T-200 (Ly 5), the reaction of the B cell tumors reflected the expression of these markers on comparable normal cells. This investigation also emphasized the marked intratumor and intertumor heterogeneity that is observed when cloned cell lines are analyzed quantitatively for a large number of surface markers. Thus, this approach may be particularly useful in defining heterogeneity in maturation states within cloned tumor cell lines.
Tokuhisa, T., Gadus, F. T. and Herzenberg, L. A. (1981). “Monoclonal antibody to an IgD allotype induces a new type of allotype suppression in the mouse.” J. Exp. Med. 154(3): 921-934.
Studies presented here show that perinatal exposure to anti-IgD allotype antibodies induces a persistant IgG-allotype suppression in the mouse that differs markedly from either the short-term or chronic allotype suppressions induced by antibodies to IgG or IgM allotypes. This novel form of allotype suppression induced by injecting neonatal BALB/c x SJL mice with monoclonal antibody to the paternal Igh-5b (IgD) allotype drastically reduces paternal allotype production during the first 6 mo of the affected animal's life and simultaneously stimulates compensatory production of maternal allotype IgG. In addition, it interferes with the development of B cells carrying the paternal IgD allotype and impairs the development of memory B cells destined to give rise to paternal allotype IgG-producing cells. Thus, its properties make it more like allotype suppression as described in the rabbit than like the known forms of allotype suppression in the mouse. As shown here, Igh-5b-bearing (5b+) B cells are completely depleted from the neonate after anti-5b exposure and only gradually appear as the animal ages. The recovery of the 5b+ population to near normal size (by approximately 14 wk of age) substantially preceeds recovery of the ability to generate normal-size memory B cell populations. Paternal allotype levels in serum remain well below normal until the anti-5b-exposed animals reach approximately 6 mo of age and then climb rapidly, finally stabilizing at levels comparable to levels in controls of the same age. The elevated maternal allotype levels characteristic of the suppression period began falling somewhat earlier and are clearly stabilized within the normal range in 6-mo-old animals. Thus, perinatal exposure to anti-5b compromises B cell development and IgG production throughout early adulthood but has little apparent effect in older animals. Perinatal exposure to antibody to the paternal IgG2a allotype (Igh-1b) or IgM allotype (Igh-6b), in contrast, induces a chronic allotype suppression that has relatively little affect on IgG production in young adults but severely suppresses allotype production in older animals. Furthermore, this type of (chronic) suppression does not influence maternal allotype production and does not interfere with memory B cell development. These differences, illustrated here by data from parallel sets of animals exposed either toi anti-5b or anti-1b, raise a series of intriguing questions concerning the mechanisms regulating B cell development and expression and the nature of the neonatal (B) cell populations with which the suppression-inducing antibodies react.
Ledbetter, J., Tsu TT, Herzenberg LA (1981). Differentiation antigens on spontaneous and transplanted AKR leukemias. Hybridomas in the Diagnosis and Treatment of Cancer. O. MItchell MS.
Parsons, M., Cazenave, P. A. and Herzenberg, L. A. (1981). “Igh-4D, a new allotype at the mouse IgG1 heavy chain locus.” Immunogenetics 14(3-4): 341-4.
Reidler, J., Oi, V. T., Carlsen, W., Vuong, T. M., Pecht, I., Herzenberg, L. A. and Stryer, L. (1982). “Rotational dynamics of monoclonal anti-dansyl immunoglobulins.” Journal of Molecular Biology 158(4): 739-46.
Bakhshi, A., Miyasaka, N., Kavathas, P., Daniels, T. E., Strand, C. V., Herzenberg, L. A. and Talal, N. (1983). “Lymphocyte subsets in Sjogren's syndrome: a quantitative analysis using monoclonal antibodies and the fluorescence-activated cell sorter.” Journal of Clinical & Laboratory Immunology 10(2): 63-9.
Lymphocyte subsets in the peripheral blood of 18 patients with Sjogren's syndrome (SS) were studied using monoclonal antibodies and the fluorescence-activated cell sorter (FACS). The percentage of T cells was decreased when compared to normal controls. In primary SS, there was a proportional decrease in both suppressor/cytotoxic (anti-Leu-2a reactive) and helper/inducer (anti-Leu-3a reactive) T cells with an unchanged helper/suppressor ratio (1.8 vs. 1.7 for normals). In SS with an associated connective tissue disorder, there was a significant decrease only in the suppressor/cytotoxic subset. There was increase in B cells and null cells in primary SS compared to controls. Quantitative immunofluorescence allowed the calculation of determinant density per cell. Cells expressing low antigen density Leu-2a were increased in 8 patients (4 with primary SS and 4 with SS with an associated disorder). Thus, in addition to quantitative changes in lymphocyte subsets, we found changes in Leu-2a expression suggesting abnormal differentiation of the suppressor/cytotoxic subset. These changes may contribute to the immunoregulatory disturbance in Sjogren's syndrome.
Huber, B. T., Tokuhisa, T. and Herzenberg, L. A. (1981). “Primary and secondary in situ antibody response: abnormal affinity maturation pattern in mice carrying the xid gene.” European Journal of Immunology 11(5): 353-7.
The xid gene in mice controls a recessive defect resulting in the absence of a late maturing subset of B cells. Whereas the responsiveness pattern of these mice have been clearly defined in terms of their ability or inability to make antibodies to certain classes of thymus-independent antigens, there are conflicting reports in regard to affinity maturation of the antibody response to thymus-dependent antigens. To resolve this controversial issue, the two major isotypes of the IgG response, namely IgG1 and IgG2a were examined with a highly sensitive radioimmunoassay that measures both the magnitude and affinity of the anti-2,4-dinitrophenyl antibody of each isotype in individual serum samples. It was found that the xid gene reduced the amount but not affinity of the IgG1 antibody produced, whereas it impaired the whole IgG2a responses severely. In fact, mice carrying the defective gene were unable to mount a secondary IgG2a response, measured either quantitatively or qualitatively in terms of increased affinity. To test the possibility that Lyb3, an isogenic B cell-triggering receptor lacking in xid-mutant mice, plays a direct role in the maturation of the immune response, the antibody profile in normal mice immunized eigher with antigen alone or in combination with anti-Lyb3 receptor substantially elevated and accelerated the primary IgG2a response, whereas it had little effect on the IgG1 response.
Herzenberg, L. A., T. Tokuhisa, L.A. Herzenberg (1981). Carrier specific suppression operates through the hapten-specific system. Immunoglobulin Idiotypes and theit Expression, INC-UCLA Symposia on Molecular and Cellular Biology. XX.