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Herzenberg, L. A., Stall, A. M., Braun, J., Weaver, D., Baltimore, D., Herzenberg, L. A. and Grosschedl, R. (1987). “Development of the predominant B cell lineage is imparied in immunoglobulin heavy chain transgenic mice.” Nature 329: 71-73.
Waldor, M. K., Mitchell, D., Kipps, T. J., Herzenberg, L. A. and Steinman, L. (1987). “Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody.” Journal of Immunology 139(11): 3660-4.
Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.
Panka, D. J., Mudgett-Hunter, M., Parks, D. R., Peterson, L. L., Herzenberg, L. A., Haber, E. and Margolies, M. N. (1988). “Variable region framework differences result in decreased or increased affinity of variant anti-digoxin antibodies.” Proceedings of the National Academy of Sciences of the United States of America 85(9): 3080-4.
Rare spontaneous variants of the anti-digoxin antibody-producing hybridoma 40-150 (Ko = 5.4 x 10(9) M-1) were selected for altered antigen binding by two-color fluorescence-activated cell sorting. The parent antibody binds digoxin 890-fold greater than digitoxin. The variant 40-150 A2.4 has reduced affinity for digoxin (Ko = 9.2 x 10(6) M-1) and binds digoxin 33-fold greater than digitoxin. A second-order variant, derived from 40-150 A2.4 (designated 40-150 A2.4 P.10), demonstrated partial regain of digoxin binding (Ko = 4.4 x 10(8) M-1). The altered binding of the variant 40-150 A2.4 was accounted for by a point mutation resulting in substitution of arginine for serine at position 94 in the heavy chain variable region. Antibody 40-150 A2.4 P.10 also contains this arginine but owes its enhanced antigen binding to deletion of two amino acids from the heavy chain amino terminus. This unusual sequence alteration in an immunoglobulin framework region confers increased affinity for antigen.
Nolan, G. P., Fiering, S., Nicolas, J. F. and Herzenberg, L. A. (1988). “Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.” Proceedings of the National Academy of Sciences of the United States of America 85(8): 2603-7.
We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.
Stall, A. M., Kroese, F. G., Gadus, F. T., Sieckmann, D. G. and Herzenberg, L. A. (1988). “Rearrangement and expression of endogenous immunoglobulin genes occur in many murine B cells expressing transgenic membrane IgM.” Proceedings of the National Academy of Sciences of the United States of America 85(10): 3546-50.
Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).
Nakauchi, H., Nolan, G. P., Tagawa, M. and Herzenberg, L. A. (1987). Cloning, sequencing and differential splicing of the Lyt-2 gene. New Horizons in Animal Models for Autoimmune Disease, Academic Press: 133-140.
Dangl, J. L., Wensel, T. G., Morrison, S. L., Stryer, L., Herzenberg, L. A. and Oi, V. T. (1988). “Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies.” EMBO Journal 7(7): 1989-94.
We generated a family of chimeric immunoglobulin G (IgG) molecules having identical antigen-combining sites for the dansyl (DNS) hapten, in conjunction with nine heavy chain constant (CH) regions. This family of antibody molecules allows comparison of CH dependent properties independent of possible variable region contributions to IgG function. The segmental flexibility and complement fixation activity were measured of six genetically engineered molecules (the four human IgG isotypes, mouse IgG3 and rabbit IgG) and the remaining three mouse IgG isotypes, (IgG1, IgG2a and IgG2b), isolated previously by somatic cell genetic techniques. These properties of antibody molecules each correlate with the length of the immunoglobulin hinge region which separate the first and second CH (CH1 and CH2) domains. These results attribute a structural basis for two critical properties of antibody molecules.
Stall, A. M., Farinas, M. C., Tarlinton, D. M., Lalor, P. A., Herzenberg, L. A. and Strober, S. (1988). “Ly-1 B-cell clones similar to human chronic lymphocytic leukemias routinely develop in older normal mice and young autoimmune (New Zealand Black-related) animals.” Proceedings of the National Academy of Sciences of the United States of America 85(19): 7312-6.
Studies presented here demonstrate that individually expanded clones of murine Ly-1 B cells, perhaps analogous to the expanded neoplastic Leu-1 B-cell clones in human chronic lymphocytic leukemias, are universally detectable in young New Zealand Black (NZB)-related autoimmune mice and in senescent normal mice (greater than 18 months old). These clones are visible as phenotypically homogeneous cell populations in multiparameter fluorescence-activated cell sorter analyses of peritoneal and splenic B cells; they show unique immunoglobulin heavy- and light-chain gene rearrangements in Southern gel analyses of peritoneal and splenic DNA; and, like the self-replenishing Ly-1 B-cell population from which they are drawn, they tend to grow readily in irradiated or unirradiated syngeneic or allotype congenic hosts. Furthermore, they develop and generalize in primary and secondary hosts in a characteristic pattern (peritoneum much greater than spleen greater than lymph node greater than bone marrow) that suggests that their initial growth is controlled by the mechanisms that normally control Ly-1 B-cell distribution in lymphoid organs. The universal emergence of these clones within the Ly-1 B-cell lineage may be explained by the substantially greater opportunity for hyperplastic and neoplastic transformation events in this long-lived self-replenishing Ly-1 B-cell population, which must divide relatively frequently to maintain its normal size throughout adulthood. Repeated exposure to internal or environmental antigens (with which Ly-1 B cells are known to react) may also play a role in driving the development of these clones.
LAH #30
Kroese, F. G., Butcher, E. C., Stall, A. M. and Herzenberg, L. A. (1989). “A major peritoneal reservoir of precursors for intestinal IgA plasma cells.” Immunological Investigations 18(1-4): 47-58.
Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyer's patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.
Alberti, S. and Herzenberg, L. A. (1988). “DNA methylation prevents transfection of genes for specific surface antigens.” Proceedings of the National Academy of Sciences of the United States of America 85(22): 8391-4.
Sperm and trophoblast are among the few nucleated human cells that do not express HLA class I antigens. DNA methylation, which is proposed to be a tight mechanism of regulation, may be necessary to turn off these genes. We have investigated the transfectability of HLA class I genes and of the genes for the T-cell differentiation antigens Leu-1 (CD5) and Leu-2 (CD8) in mouse L cells by using human sperm cells and choriocarcinoma cell lines, tumors of trophoblastic origin, as sources of DNA. It was found that DNA from one choriocarcinoma line (JAR) does not transfect genes for HLA, Leu-1, or Leu-2 and that DNA from two other choriocarcinoma lines (BeWo and Ima) transfects only some of the surface markers. Sperm DNA transfects genes for all the surface antigens tested except Leu-1. DNA from control cells and from the line SCH transfects all the markers studied. Southern blots show that all cell types contain apparently intact genes encoding HLA, Leu-1, and Leu-2 and reveal differences in the DNA methylation patterns of genes from different sources of DNA. We treated JAR (the cell line with the lowest transfecting ability) with 5-azacytidine and obtained demethylation of its DNA. This demethylated DNA transfects genes for both HLA class I antigens and Leu-2. Further culture of JAR cells in the absence of 5-azacytidine results in remethylation of their DNA and decreased ability to transfect these surface antigens. These findings indicate that DNA methylation affects the efficiency of transfection of surface antigen genes in L cells.
Tarlinton, D., Stall, A. M. and Herzenberg, L. A. (1988). “Repetitive usage of immunoglobulin VH and D gene segments in CD5+ Ly-1 B clones of (NZB x NZW)F1 mice.” EMBO Journal 7(12): 3705-10.
The usually small Ly-1 B cell population is markedly increased in older mice by expansion of certain clones. This results in a cellular picture very similar to human B chronic lymphocytic leukemia. Here we report a molecular analysis of the immunoglobulin gene rearrangements of the Ly-1 B cell populations in (NZB x NZW)F1 females. We find that (i) the number of clones found in the peritoneum (a major tissue source of Ly-1 B cells) decreases with age till mono- or biclonality is common by approximately 6 months, (ii) many clones from different mice show the same size rearrangements at both the Ig heavy and light chain loci and (iii) the IgH rearrangements found in a clone isolated from the spleen of one mouse are a subset of those found in the peritoneum of the same mouse, implying migration occurs from the peritoneum to the spleen. Molecular cloning and sequencing of the IgH rearrangements from the peritoneal clones of one B/W mouse revealed that all productive rearrangements used the identical unmutated VH and D elements joined to different JHS. Indeed, two VDJH4 rearrangements were recovered which were identical but for six junctional (N region) nucleotides. The conservation of VH and D segment usage in the rearrangements of these Ly-1 B cell clones could indicate some strong selective pressure for clonal expansion (for example antigen selection) operates via the immunoglobulin molecules of these cells. Southern analyses of other (NZB x NZW)F1 mice with this cloned VH and the usage of the same or similar VH genes among a number of Ly-1 B origin tumors in other mouse strains indicate the generality of this repetitive VH gene usage in individual mice.
Kerr, W. G., Nolan, G. P. and Herzenberg, L. A. (1991). In situ detection of transcriptionally-active chromatin and genetic regulatory elements in individual viable mammalian cells. Mechanisms of Lymphocyte Activation and Immune Regulation III. S. e. a. Gupta. New York, Plenum Press: 187-200.
Kerr, W. G., Nolan, G. P. and Herzenberg, L. A. (1989). “In situ detection of transcriptionally active chromatin and genetic regulatory elements in individual viable mammalian cells.” Immunology. Supplement 2: 74-8; discussion 79.
Using a newly developed FACS method for quantifying the expression of the Escherischia coli lacZ reporter gene in viable mammalian cells, we have obtained cloned cell lines in which the expression of lacZ is under the control of native endogenous transcription elements. We infected the murine pre-B cell 70Z/3 with transcriptionally disabled retroviruses containing lacZ and employed the FACS-FDG technique to detect and sort rare lacZ+ cells in which we expect integration is near such endogenous transcription elements. After two rounds of enrichment we obtained a population of cells that was 80-90% positive for lacZ activity. Clones derived from the lacZ+ pool differ from each other with respect to their overall level of lacZ activity as well as in the pattern of lacZ expression among cells within an individual clone. Treatment of these lacZ+ 70Z/3 clones with lipopolysaccharide (LPS; which is known to stimulate differentiation of 70Z/3 from a pre-B cell to an IgM-expressing B cell) greatly decreased lacZ expression in one clone, 7e17. lacZ expression in this clone was 50-100 times lower within 24 hr of LPS addition and coincided with the acquisition of IgM kappa on the surface of 7e17. This suggests that a transcriptionally active domain of chromatin that harbors the lacZ construct is down-regulated during the transition induced by LPS stimulation.
Lalor, P. A., Stall, A. M., Adams, S. and Herzenberg, L. A. (1989). “Permanent alteration of the murine Ly-1 B repertoire due to selective depletion of Ly-1 B cells in neonatal animals.” European Journal of Immunology 19(3): 501-6.
Studies presented here demonstrate that paternal allotype Ly-1 B cells are permanently depleted following neonatal treatment with antibodies to the paternal IgM allotype. Paternal allotype conventional B cells, in contrast, are temporarily depleted by treatment with either anti-IgM or anti-IgD allotype antibodies and return rapidly to normal frequencies once the antibody treatment disappears. These differences are explained by basic developmental differences between Ly-1 B and conventional lineage B cells. That is, the conventional B cell population is replenished from Ig- precursors throughout life and, therefore, is only temporarily affected when depleted in neonates. The Ly-1 B cell population, in contrast, develops from Ig- progenitors during the prenatal and neonatal life but survives because it is exclusively self-replenishing in adults. Therefore, elimination of a population of Ly-1 B cells from neonates is tantamount to removing it forever. These findings suggest that while conventional B cells turn over rapidly and have an effectively unlimited repertoire, Ly-1 B cells express a repertoire whose composition is strongly influenced by neonatal conditions that favor or select against the retention of cells producing certain antibody molecules. Thus, Ly-1 B cells play a unique role in the immune system in that they retain indefinitely the history of the neonatal animal's immunological experience.
Lalor, P. A., Herzenberg, L. A., Adams, S. and Stall, A. M. (1989). “Feedback regulation of murine Ly-1 B cell development.” European Journal of Immunology 19(3): 507-13.
Studies presented here, conducted with allotype homozygotes, demonstrate the existence of a feedback mechanism that regulates development of Ly-1 B cells from immature progenitors. In the preceding study (P. A. Lalor et al., Eur. J. Immunol. 1989. 19:501), conducted with allotype heterozygotes, we showed that treating neonates with monoclonal antibody to the paternal allotype IgM depletes roughly half of the neonatal B cell population (i.e. those expressing the paternal IgM allotype) and that paternal allotype Ly-1 B cells specificically remain depleted for the life of the animal. Here we show that treating allotype homozygotes with the same antibody depletes all (rather than half) of the B cells and that, under these conditions, relatively normal numbers of Ly-1 B cells reappear shortly after the treatment antibody disappears. The recovery, we also show, is prevented by restoring allotype-congenic Ly-1 B cells to the treated homozygotes, i.e. by reconstituting treated neonates with allotype-congenic peritoneal cells, sorted Ly-1 B cells or a monoclonal population of Ly-1 B "tumor" cells. These findings in essence reveal a feedback mechanism through which mature Ly-1 B cells prevent further Ly-1 B cell development from Ig- precursors. This feedback regulation is independent of Ig secretion by the mature Ly-1 B cells, since the monoclonal Ly-1 B "tumor" population that prevents endogenous Ly-1 B development does not secrete Ig. Furthermore, it appears to be independent of Ly-1 B surface Ig specificity, since a monoclonal population is sufficient to block all Ly-1 B cell development. This mechanism appears to operate normally to fix the composition of the Ly-1 B population, which survives through self-replenishment in adults, in accord with conditions that influence Ly-1 B development during neonatal life.
Kroese, F. G., Butcher, E. C., Stall, A. M., Lalor, P. A., Adams, S. and Herzenberg, L. A. (1989). “Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.” International Immunology 1(1): 75-84.
Long term B lineage chimeras are used here to study the origin of plasma cells in the mouse. Chimeric mice are constructed by reconstituting lethally irradiated mice with peritoneal cells (PerC) and bone marrow cells from congenic pairs of mice differing in Igh-C allotype. All conventional B cells in these mice express the allotype of the bone marrow donor and nearly all Ly-1 B lineage cells express the allotype of the PerC donor. FACS analysis and immunohistology of these mice shows that virtually all (sig+) B cells in peripheral lymphoid organs are derived from the bone marrow donor. However, despite this overwhelming number of bone marrow-derived B cells in these animals, immunohistological staining of lymphoid organs and gut shows that nearly half of the IgM, IgG, and IgA plasma cells derive from the PerC donor. These data demonstrate that the peritoneal cavity contains a major reservoir of self-replenishing cells that play a significant role in the mucosal immune response. The possibility that these are B cells that belong to the Ly-1 B lineage is discussed.
Parks, D. R., Herzenberg, L. A. and Herzenberg, L. A. (1989). Flow cytometry and fluorescence-activated cell sorting. Fundamental Immunology. W. E. Paul. New York, Raven Press: 781-802.
Herzenberg, L. A., Lalor, P. A. and Stall, A. M. (1989). “Are Ly-1 B cells important in autoimmune disease?” Journal of Autoimmunity 2 Suppl: 225-31.
Although Ly-1 B cells produce autoantibodies and are found at elevated frequencies in certain autoimmune strains, very little is known about the role of these cells, if any, in autoimmune disease. In this publication, we summarize some recent findings relevant to Ly-1 B-cell development, clonal expansion and antibody production. We then consider the idea that Ly-1 B cells constitute the most primitive B-cell lineage in mammals and that the evolutionary niche occupied by these cells requires that they produce a basic set of autoantibodies that cross-react with common bacterial antigens.
Yancopoulos, G. D., Nolan, G. P., Pollock, R., Prockop, S., Li, S. C., Herzenberg, L. A. and Alt, F. W. (1989). Lymphoid VDJ recombinase activity: Development of a novel fluorescence-based assay system. Vectors as Tools for the Study ofNormal and Abnormal Growth and Differentiation. H. e. a. Lother. Berlin and Heidelberg, Springer Verlag. 34.
Jutila, M. A., Kroese, F. G., Jutila, K. L., Stall, A. M., Fiering, S., Herzenberg, L. A., Berg, E. L. and Butcher, E. C. (1988). “Ly-6C is a monocyte/macrophage and endothelial cell differentiation antigen regulated by interferon-gamma.” European Journal of Immunology 18(11): 1819-26.
Using a new Ly-6C-specific antibody (Monts-1) we show that this class of antigens are differentially expressed on monocytes/macrophages and endothelial cells. Recently elicited peritoneal exudate Mac-1+ mononuclear cells, as well as Mac-1+ mononuclear cells in the bone marrow and in the peripheral blood, express high levels of Ly-6C. Ly-6C+ mononuclear Mac-1+ cells are absent in normal uninflamed skin, but are present in high numbers in skin lesions 3 days after the s.c. injection of lipopolysaccharide, concanavalin A or complete Freund's adjuvant. In addition, large Ly-6C+ mononuclear cells are predominant in chronic granulomas induced by complete Freund's adjuvant. Resident macrophages in a variety of tissues express low levels or in many cases do not express Ly-6C. Two out of three monocyte-like cell lines are Ly-6C+, whereas macrophage-like cell lines are negative. Ly-6C+ monocytes/macrophages lose the Ly-6C antigen within 24 h after in vitro culture. Ly-6C- cultured monocytes and Ly-6C- monocyte-like cell lines, but not fully differentiated macrophages and macrophage-like cell lines, can be induced to express the Ly-6C antigen by interferon-gamma. A population of small vessel endothelial cells in diverse tissues also express high levels of Ly-6C. The present findings suggest that the Ly-6C antigen family, shown by others to be involved in T cell activation, may have more general importance in immune responses and cellular differentiation than previously appreciated.
Herzenberg, L. A. (1989). “Toward a layered immune system.” Cell 59(6): 953-4.
Leclerc, C. and Herzenberg, L. A. (1989). “Regulation of antibody production by suppressor T cells.” Res. Immunol 140: 285-345.
LAH #321-01
Herzenberg, L. A. (1989). “Murine suppressor T cells: mirage or cloudy reality?” Research in Immunology 140(3): 337-8; discussion 339-45.
Kerr, W. G., Nolan, G. P., Serafini, A. T. and Herzenberg, L. A. (1989). “Transcriptionally defective retroviruses containing lacZ for the in situ detection of endogenous genes and developmentally regulated chromatin.” Cold Spring Harbor Symposia on Quantitative Biology 54 Pt 2: 767-76.
Herzenberg, L. A. and Stall, A. M. (1989). “Conventional and Ly-1 B-cell lineages in normal and mu transgenic mice.” Cold Spring Harbor Symposia on Quantitative Biology 54 Pt 1: 219-25.
LAH #324
Herzenberg, L. A. and Stall, A. (1989). B cell lineages and immunologic memory. Progress in Immunology VIII. F. Melchers.
Yancopoulos, G. D., Nolan, G. P., Pollock, R., Prockop, S., Li, S. C., Herzenberg, L. A. and Alt, F. W. (1990). “A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity.” Molecular & Cellular Biology 10(4): 1697-704.
We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector.(ABSTRACT TRUNCATED AT 250 WORDS)
LAH #326
Farinas, M. C., Stall, A. M., Solovera, J. J., Tarlinton, D. M., Herzenberg, L. A. and Strober, S. (1990). “Ly-1 B cells and disease activity in (New Zealand black x New Zealand white)F1 mice. Effect of total lymphoid irradiation.” Arthritis & Rheumatism 33(4): 553-62.
The treatment of female (New Zealand black x New Zealand white)F1 mice with total lymphoid irradiation resulted in a prolonged remission of autoimmune disease activity. Total lymphoid irradiation-treated mice also showed a marked reduction of Ly-1 B cells, which lasted up to 3 months. The subsequent return of Ly-1 B cells to preirradiation levels was not associated with a simultaneous return of disease when measured by parameters such as IgG anti-DNA antibodies and spontaneous secretion of IgG by splenic cells. In cell sorting experiments, most of the cells spontaneously secreting IgG were found within the Ly-1- (CD5-) splenic B cell population.
Portnoi, D., Stall, A. M., Schwartz, D., Merigan, T. C., Herzenberg, L. A. and Basham, T. (1990). “Zidovudine (azido dideoxythymidine) inhibits characteristic early alterations of lymphoid cell populations in retrovirus-induced murine AIDS.” Journal of Immunology 144(5): 1705-10.
Using flow cytometry technology and multiparameter analyses, we report early and characteristic alterations in lymphoid cell profile in spleen and lymph nodes due to LP-BM5 retrovirus disease (murine AIDS (MAIDS)) and the effect of azido dideoxythymidine, a nucleoside inhibitor, on these changes. MAIDS has been characterized by rapid and profound lymphoproliferation accompanied by hypergammaglobulinemia and immunosuppression. As early as 2 wk postinfection, there is a selective depletion of CD8+ cells whereas the total number of CD4+ cells increases throughout the first 8 wk of infection although the frequency is relatively stable. These population changes were partially delayed by oral AZT therapy for 6 wk postinfection. Ly-6C (AL-21) is expressed on roughly 50% of CD4+ and CD8+ cells in C57BL/6 mice. In MAIDS, the residual population of CD8+ cells is primarily Ly-6C+. The CD4+ cells have a transient increase in ratio of Ly-6C+/Ly-6C- cells at 2 wk postinfection but by 6 wk are primarily Ly-6C-. There was an increase in both the total number and percentage of Mac 1+ cells and a selective depletion of certain splenic B cell subpopulations. Azido dideoxythymidine delays these early population changes.
Fiering, S., Northrop, J. P., Nolan, G. P., Mattila, P. S., Crabtree, G. R. and Herzenberg, L. A. (1990). “Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor.” Genes & Development 4(10): 1823-34.
Stimulation of T lymphocytes through their antigen receptor leads to the appearance of several transcription factors, including NF-AT and NF-kappa B, which are involved in regulating genes required for immunologic activation. To investigate the activity of a single transcription factor in individual viable cells, we have applied an assay that uses the fluorescence-activated cell sorter to quantitate beta-galactosidase (beta-gal). We have analyzed the distribution of NF-AT transcriptional activity among T cells undergoing activation by using a construct in which three tandem copies of the NF-AT-binding site directs transcription of the lacZ gene. Unexpectedly, stimulation of cloned stably transfected Jurkat T cells leads to a bimodal pattern of beta-gal expression in which some cells express no beta-gal and others express high levels. This expression pattern cannot be accounted for by cell-cycle position or heritable variation. Further results, in which beta-gal activity is correlated with NF-AT-binding activity, indicate that the concentration of NF-AT must exceed a critical threshold before transcription initiates. This threshold likely reflects the NF-AT concentration-dependent assembly of transcription complexes at the promoter. Similar constructs controlled by NF-kappa B or the entire interleukin-2 enhancer show bimodal expression patterns during induction, suggesting that thresholds set by the concentration of transcription factors may be a common property of inducible genes.
Kerr, W. G., Nolan, G. P., Johnsen, J. B. and Herzenberg, L. A. (1991). “In situ detection of stage-specific genes and enhancers in B cell differentiation via gene-search retroviruses.” Advances in Experimental Medicine & Biology 292: 187-200.
We demonstrate that infection of an LPS-responsive pre-B cell line with transcriptionally-defective retroviruses containing a reporter gene (lacZ) can result in viral integrations where expression of lacZ is differentiation stage-dependent. Because expression of lacZ is dependent upon flanking cellular sequences these retroviral integrations represent in situ gene fusions with cellular enhancers (Enhsr1) and genes (Gensr1) which are either induced or repressed during LPS-stimulated differentiation. One of the well-documented effects of LPS upon pre-B cells is the induction of kappa light chain transcription via NF-kappa B. The identification of LPS-stimulated gene repression during B cell differentiation indicates that LPS has multiple effects upon gene expression during the pre-B to B cell transition. The identification of cellular enhancers and genes which are downregulated during the transition from the pre-B to the B cell stage indicates that other transcription factors, in addition to NF-kappa B, are required for this step in differentiation. Finally, we present some initial experiments which indicate the gene-search retroviruses can introduce expression of lacZ into normal hematopoietic cells in vitro and in vivo.
Chen, J. Z., Stall, A. M. and Herzenberg, L. A. (1990). “Differences in glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially enable transduction of different signals.” EMBO Journal 9(7): 2117-24.
Studies presented here demonstrate that IgM and IgD molecules on normal murine B lymphocytes exist in different, noncovalently associated molecular complexes containing distinct but potentially related glycoproteins. The glycoproteins in these complexes, particularly those associated with IgD, show striking differences in various lymphoid organs and in X-linked immunodeficient (Xid) mice. These differences are due in part to post-translational processing. They apparently reflect the differential expression of the Ig-associated glycoproteins in the various B cell subpopulations and lineages and the differential distribution of the subpopulations and lineages in the various lymphoid organs. In addition, they reflect structural differences in the IgM and IgD complexes which, we suggest, permit differential signal transduction by IgM and IgD on the same B cell.
Kipps, T. J., Parham, P., Punt, J. and Herzenberg, L. A. (1985). “Importance of immunoglobulin isotype in human antibody-dependent, cell-mediated cytotoxicity directed by murine monoclonal antibodies.” Journal of Experimental Medicine 161(1): 1-17.
Using the fluorescence activated cell sorter to select rare IgG2a- and IgG2b-producing variants, we developed switch variant families of hybridomas from IgG1-producing hybridomas, ME1 and MA2.1. The IgG2a and IgG2b antibodies produced by such switch variants have the same binding activities for HLA as the IgG1 antibodies produced by the parent hybridomas. Using these antibodies, we directly compared the IgG1, IgG2a, and IgG2b murine Ig isotypes for their capacities to direct human peripheral blood lymphocytes (PBL) in antibody-dependent cell-mediated cytotoxicity (ADCC) against a B lymphoblastoid cell line. We demonstrate that, for antibodies of identical binding affinity and specificity, the murine IgG2a isotype is the most effective in directing ADCC by human effector cells. The murine IgG2b directs intermediate levels of ADCC activity while IgG1 is inactive. We identified the effector cells in human PBL that mediate IgG2a or IgG2b ADCC as nonadherent killer (K) cells. These cells express the C3bi receptor and have cytolytic activity which is specifically blocked by a monoclonal antibody (anti-Leu-11a) that binds the Fc receptor (FcR) of such cells. Finally, FcR-bearing K cells bind to target cell-bound, rather than free, IgG2a or IgG2b molecules.
Chen, J. and Herzenberg, L. A. (1991). “Heparin alters the expression of different forms of immunoglobulin mu heavy chains and their associated proteins by pre-B cell lines and normal Ly-1 (CD5+) B cells.” International Immunology 3(11): 1117-27.
Studies presented here show that heparin alters immunoglobulin expression by murine pre-B cell lines and normal Ly-1 (CD5+) B cells. Previous studies have shown that pre-B cell lines 70Z/3 and NFS-5.3 express mu heavy chains in the cytoplasm and a small amount on the cell surface. Both these cytoplasmic and surface mu are disulfide-linked to omega (lambda 5) surrogate light chains and are noncovalently associated with iota (Vpre-B) variable region-like proteins. We show that culturing 70Z/3 with heparin reduces the amount of the membrane-form mu (micron) on the cell surface. Culturing NFS-5.3 with heparin similarly decreases the membrane-form mu; however, it increases the surface level of a pentameric mu molecule containing secreted-form mu (microS) heavy chains, disulfide-linked omega (lambda 5) chains, and noncovalently associated proteins. Culturing peritoneal B cells with heparin also increases the production of the secreted-form microS, detectable in this case by the secretion of classical pentameric IgM. Similarly, injecting heparin intraperitoneally increases IgM secretion by peritoneal Ly-1 B cells. Thus heparin could influence pre-B cell and B cell differentiation and function.
LAH #332
Kroese, F. G., Butcher, E. C., Lalor, P. A., Stall, A. M. and Herzenberg, L. A. (1990). “The rat B cell system: the anatomical localization of flow cytometry-defined B cell subpopulations.” European Journal of Immunology 20(7): 1527-34.
Two-color flow cytometrical (FCM) analysis of rat peripheral lymphoid organs shows two distinct IgM/IgD-defined B cell subpopulations, similar to those of the mouse: a major population of cells expressing little IgM and high levels of IgD (population I) and a minor population of cells expressing high levels of IgM but little IgD (population III). In peripheral lymphoid organs population III cells are mainly found in spleen where they represent about 25% of the B cells; population III cells are almost absent from lymph nodes and Peyer's patches. In adult bone marrow and in neonatal spleen the majority of IgM/IgD-defined B cells (greater than 70%) are population III cells, similar to what is observed in the mouse. In contrast with mice, only a low proportion of the cells (1%) recovered from the peritoneal cavity are B cells, and most of them belong to population I. Previously defined monoclonal antibodies (HIS22 and HIS24) to B cell forms of the leukocyte common antigen (CD45R) in combination with staining for surface IgM and surface IgD demonstrates a further heterogeneity of rat B cells by three-color FCM analyses. HIS22 labels most population I cells; population III cells and a small subset (about one third) of population I express only very low levels of the HIS22 determinant. HIS24 reacts with population I cells and subdivides population III into two subsets: about one third of splenic population III cells are brightly stained with this antibody whereas fluorescence of the remaining two-thirds is lower. The HIS24bright population III cells likely are newly formed B cells since cells with this phenotype are the predominant surface Ig population found in adult bone marrow and neonatal spleen. In tissue sections of lymphoid organs, HIS22- and HIS24-positive cells are mainly found in lymphoid follicles; splenic marginal zones are almost unstained. Combining immunohistological analysis with the FCM data, we therefore conclude that the small follicular B cells are in population I and marginal zone B cells are found in the HIS24dull population III. The in situ localization of HIS24bright population III cells and the HIS22dull population I cells is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)
Roederer, M., Staal, F. J., Raju, P. A., Ela, S. W. and Herzenberg, L. A. (1990). “Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine.” Proceedings of the National Academy of Sciences of the United States of America 87(12): 4884-8.
We show that the stimulation of human immunodeficiency virus (HIV) brought about by tumor necrosis factor alpha and phorbol 12-myristate 13-acetate can be inhibited by adding N-acetyl-L-cysteine (NAC). NAC, which replenishes intracellular glutathione, effectively inhibits the tumor necrosis factor alpha- or phorbol ester-stimulated replication of HIV in acutely infected cell cultures. NAC also inhibits the cytokine-enhanced HIV long terminal repeat-directed expression of beta-galactosidase in in vitro HIV model systems. These results show that intracellular thiol levels influence HIV production. Furthermore, because NAC reverses tumor necrosis factor alpha toxicity both in cells and in animals and is a well-known drug that can be administered orally without known toxicity in humans, these results suggest that NAC is a possible therapeutic agent in AIDS.
Nawata, Y., Stall, A. M., Herzenberg, L. A., Eugui, E. M. and Allison, A. C. (1990). “Surface immunoglobulin ligands and cytokines differentially affect proliferation and antibody production by human CD5+ and CD5- B lymphocytes.” International Immunology 2(7): 603-14.
Normal human peripheral blood B lymphocytes were separated into CD19+ CD5+ and CD19+ CD5- subsets by dual-color FACS sorting. In most experiments the cells were activated with Staphylococcus aureus Cowan I (SAC) and cultured in the absence or presence of recombinant human IL-1 alpha, IL-2, or IL-6, or combinations of these cytokines. Unstimulated CD5+ and CD5- B cells showed a comparable, low level of incorporation of [3H]thymidine into DNA. SAC stimulated proliferation of CD5+ and CD5- B cells, and this proliferation was augmented by IL-2 in the case of CD5- B cells. Anti-mu beads stimulated some proliferation of the CD5- subset and augmented SAC-induced proliferation of these cells. In contrast, anti-mu beads did not stimulate proliferation of the CD5+ subset and had no effect on SAC-induced proliferation of these cells. CD5+ B cells activated by anti-mu beads were stimulated to proliferate in the presence of IL-4, but not in the presence of IL-2. These observations support the interpretation that two signals are required for proliferation of CD5+ B cells. Using a two-step culture system, SAC activation itself did not induce Ig production by either subset of purified B cells. However, it primed the cells for antibody production in the presence of IL-2. IL-1 and IL-6 by themselves augmented antibody formation by these cells slightly, if at all. However, IL-6, and to a lesser extent IL-1, augmented antibody production in the presence of IL-2. Under the culture conditions used CD5- B cells produced IgM, IgG, and IgA whereas the CD5+ B cells produced almost exclusively IgM. The expression on B cells of surface activation markers was analyzed after culture for 2 days with SAC or anti-mu beads. In both subsets expression of Leu-23 and Leu-21 was increased, with some differences in intensity (Leu-23 greater in CD5+ cells, Leu-21 greater in CD5- cells). SAC increased IL-2R expression to a greater extent than anti-mu beads. In neither subset was expression of CD23 increased. These observations are discussed in the context of the possible role of the CD5+ subset of B lymphocytes as components of a system of natural immunity.
LAH #336
Weir, D. M., Herzenberg, L. A., Blackwell, C. C. and Herzenberg, L. A., Eds. (1986). The Handbook of Experimental Immunology. The Handbook of Experimental Immunology. Edinburgh, Blackwell Scientific Publications, Ltd.
MacGregor, G. R., Nolan, G. P., Fiering, S., Roederer, M. and Herzenberg, L. A. (1991). Use of E. coli lacZ (b-Galactosidase) as a reporter gene. Methods in Molecular Biology. E. J. Murray. Clifton, New Jersey, The Humana Press Inc. 7; Gene Transfer and Expression Protocols: 217-230.
Barsalou, T., Sujansky, W., Herzenberg, L. A. and Wiederhold, G. (1991). “Management of complex immunogenetics information using an enhanced relational model.” Computers & Biomedical Research 24(5): 476-98.
Flow cytometry has become a technique of paramount importance in the armamentarium of the scientist in such domains as immunogenetics. In the PENGUIN project, we are currently developing the architecture for an expert database system to facilitate the design of flow-cytometry experiments. This paper describes the core of this architecture--a methodology for managing complex biomedical information in an extended relational framework. More specifically, we exploit a semantic data model to enhance relational databases with structuring and manipulation tools that take more domain information into account and provide the user with an appropriate level of abstraction. We present specific applications of the structural model to database schema management, data retrieval and browsing, and integrity maintenance.
Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E., Haber, E. and Margolies, M. N. (1991). “Altered hapten recognition by two anti-digoxin hybridoma variants due to variable region point mutations.” Journal of Biological Chemistry 266(7): 4640-7.
Two spontaneous variants of the murine anti-digoxin antibody-producing hybridoma cell line 26-10 were isolated by two-color fluorescence-activated cell sorting on the basis of altered hapten binding. The variable region sequences of the antibodies produced by the mutant lines revealed that each contains a single amino acid change in the heavy chain second complementarity determining region. A Tyr to His change at position 50 leads to a 40-fold reduction in affinity for digoxin. A Ser to Phe mutation at position 52 results in a 300-fold reduction in affinity for digoxin. A competition assay involving 33 digoxin analogues was used to examine the specificity of hapten binding of 26-10 and the two mutant antibodies. The position 50 mutant has a distinct specificity change; it exhibits a preference for digoxin congeners containing a hydroxyl group at the steroid 12 position, whereas the 26-10 parent does not. The affinities of all three antibodies for hapten are progressively lowered by substitutions of increasing size at the digoxin steroid D ring 16 position. Although 26-10 binds digoxin and its genin form equally, 12 and 16 steroid position substitutions which lower affinity also confer a preference for a sugar at the steroid 3 position. These results suggest that position 50 contributes to specificity of the antibody and that alterations of the hapten can lead to differences in recognition, possibly through a shift in hapten orientation within the binding site.
Krasnow, M. A., Cumberledge, S., Manning, G., Herzenberg, L. A. and Nolan, G. P. (1991). “Whole animal cell sorting of Drosophila embryos.” Science 251(4989): 81-5.
Use of primary culture cells has been limited by the inability to purify most types of cells, particularly cells from early developmental stages. In whole animal cell sorting (WACS), live cells derived from animals harboring a lacZ transgene are purified according to their level of beta-galactosidase expression with a fluorogenic beta-galactosidase substrate and fluorescence-activated cell sorting. With WACS, incipient posterior compartment cells that express the engrailed gene were purified from early Drosophila embryos. Neuronal precursor cells were also purified, and they differentiated into neurons with high efficiency in culture. Because there are many lacZ strains, it may be possible to purify most types of Drosophila cells. The same approach is also applicable to other organisms for which germ-line transformation is possible.
Bierer, B. E., Mattila, P. S., Standaert, R. F., Herzenberg, L. A., Burakoff, S. J., Crabtree, G. and Schreiber, S. L. (1990). “Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin.” Proceedings of the National Academy of Sciences of the United States of America 87(23): 9231-5.
Proliferation and immunologic function of T lymphocytes are initiated by signals from the antigen receptor that are inhibited by the immunosuppressant FK506 but not by its structural analog, rapamycin. On the other hand, interleukin 2 (IL-2)-induced signals are blocked by rapamycin but not by FK506. Remarkably, these two drugs inhibit each other's actions, raising the possibility that both act by means of a common immunophilin (immunosuppressant binding protein). We find that the dissociation constant of rapamycin to the FK506 binding protein FKBP (Kd = 0.2 nM) is close to the dissociation constant of FK506 to FKBP (Kd = 0.4 nM) and to their effective biologic inhibitory concentrations. However, an excess of rapamycin is needed to revert FK506-mediated inhibition of IL-2 production, apoptosis, and transcriptional activation of NF-AT, a T-cell-specific transcription factor necessary for IL-2 gene activation. Similarly, an excess of FK506 is needed to revert rapamycin-mediated inhibition of IL-2-induced proliferation. The drug concentrations required for antagonism may be explained by the relative affinity of the drugs to, and by the abundance of, the immunophilin FKBP. FKBP has been shown to catalyze the interconversion of the cis- and trans-rotamers of the peptidyl-prolyl amide bond of peptide substrates; here we show that rapamycin, like FK506, is a potent inhibitor of the rotamase activity of FKBP (Ki = 0.2 nM). Neither FKBP binding nor inhibition of rotamase activity of FKBP alone is sufficient to explain the biologic actions of these drugs. Rather, these findings suggest that immunophilin bound to FK506 interferes with antigen receptor-induced signals, while rapamycin bound to the immunophilin interferes with IL-2-induced signals.
Mattila, P. S., Ullman, K. S., Fiering, S., Emmel, E. A., McCutcheon, M., Crabtree, G. R. and Herzenberg, L. A. (1990). “The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes.” EMBO Journal 9(13): 4425-33.
Cyclosporin A and FK506 are immunosuppressive compounds that have similar inhibitory effects on the expression of several lymphokines produced by T lymphocytes. Despite their similar effects the drugs bind to two different cytosolic protein, cyclophilin and FKBP respectively, which raises the possibility that they have different modes of action. Using constructs in which mRNA production controlled by a specific transcription factor could be readily measured we found that both cyclosporin A and FK506 completely inhibited transcription activated by NF-AT, NFIL2 A, NFIL2 B and partially inhibited transcription activated by NF kappa B. Cyclosporin A and FK506 inhibited only transcriptional activation that was dependent on Ca2+ mobilization. However, cyclosporin A and FK506 did not inhibit Ca2+ mobilization dependent expression of c-fos mRNA indicating that only a subset of signalling pathways regulated by Ca2+ is sensitive to these drugs. Furthermore, we did not observe any qualitative differences between the effect of cyclosporin A and FK506 on six different transcription factors which suggests that these drugs may interfere with the activity of a novel Ca2+ dependent step that regulates several transcription factors.
LAH #343
Kroese, F. G. M., Hermans, M. H., de Boer, N. K., Butcher, E. C. and Herzenberg, L. A. (1991). Rat B cell subsets identified by flow-cytometry and their anatomical ounterparts. Lymphatic Tissues and In Vivo Immune Responses. S. Ezine, S. Berrih-Aknin and B. Imhof. New York, Marcel Dekker.
LAH #344
Russo-Marie,
F., Roederer, M., Sager, B., Herzenberg, L. A. and Kaiser, D. (1993). "Beta-galactosidase
activity in single differentiating bacterial cells." Proceedings of the National
Academy of Sciences of the United States of America 90(17): 8194-8.
Myxococcus xanthus strains containing transcriptional fusions to lacZ were
analyzed and fractionated by differences in their levels of beta-galactosidase
expression. The fluorogenic substrate for beta-galactosidase, fluorescein
di-beta-galactopyranoside, was introduced into M. xanthus cells during a rapid
decrease in osmolarity of the medium followed by a return to isoosmolarity.
Fluorescein, the product of hydrolysis, was retained within the cells and
their viability was preserved. Fluorescence increased linearly with time and
was proportional to beta-galactosidase activity. beta-Galactosidase expression
in most fusion strains, though beginning at different phases of growth or
development, was distributed unimodally amongst cells. However, fusion strain
Tn5 lac omega 4473 was shown to be heterogeneous at 9 hr of development. It
was possible to separate physically cells that expressed beta-galactosidase
at a high level from other, still viable, cells with no expression. The approach
described here could be adapted to study differentiation in plants and animals
as well, where transcriptional fusions and fluorogenic substrates for enzyme
probes of gene expression also can be used.
Staal, F. J., Roederer, M. and Herzenberg, L. A. (1990). “Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus.” Proceedings of the National Academy of Sciences of the United States of America 87(24): 9943-7.
The activation of nuclear factor kappa B (NF-kappa B) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that intracellular thiol levels play a key role in regulating this process. That is, stimulation with tumor necrosis factor alpha and/or phorbol 12-myristate 13-acetate activates NF-kappa B and markedly decreases intracellular thiols; N-acetyl-L-cysteine, an efficient thiol source, prevents this thiol decrease and blocks the activation of NF-kappa B; and the lack of activated NF-kappa B prevents the activation of the HIV LTR and the transcription of genes under its control. These findings reveal a previously unrecognized genetic regulatory mechanism in which cytokine-induced shifts in intracellular thiol levels are crucial in the control of NF-kappa B activity and thereby influence the spectrum of genes expressed by cytokine-stimulated cells.
Roederer, M., Raju, P. A., Staal, F. J. and Herzenberg, L. A. (1991). “N-acetylcysteine inhibits latent HIV expression in chronically infected cells.” AIDS Research & Human Retroviruses 7(6): 563-7.
The progression of the human immunodeficiency virus (HIV) infection from its early latent (asymptomatic) stage to active, late-stage acquired immunodeficiency syndrome (AIDS) apparently begins with the production of inflammatory cytokines that stimulate the expression and replication of the latent virus. We have shown that N-acetylcysteine, a cysteine precursor that is converted intracellularly into glutathione, blocks cytokine-stimulated HIV replication in an acutely infected T-cell line and in acutely infected peripheral blood mononuclear cells from normal individuals. In this report, we show that N-acetylcysteine also inhibits stimulated HIV expression in chronically infected monocyte and T-cell lines which are used as models for latent infection in AIDS. Furthermore, we show that N-acetylcysteine blocks viral production in monocyte cell lines more effectively than it blocks viral production in T cells. Since monocytes are a major reservoir for HIV in infected individuals, these results suggest that N-acetylcysteine may slow the change from latency to the later stages of AIDS in HIV-infected individuals.
Fiering, S. N., Roederer, M., Nolan, G. P., Micklem, D. R., Parks, D. R. and Herzenberg, L. A. (1991). “Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.” Cytometry 12(4): 291-301.
The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.
#04997
Roederer, M., Fiering, S. and Herzenberg, L. A. (1991). “FACS-Gal: Flow cytometric analysis and sorting of cells expressing reporter gene constructs.” Methods: A Companion to Meth. Enzymol. 2(3 (June)): 248-260.
Herzenberg, L. A., Kantor, Aaron B., Herzenberg, Leonard A., Parks, David R. (1992). Flow cytometry. Encyclopedia of Immunology. I. M. Roitt and P. J. Delves. London, Academic Press. 2: 576-581.
Roederer, M., Staal, F.J.T., Raju, P.A., Herzenberg, L.A., Herzenberg, L.A. (1992). The Interrelationship of Tumor Necrosis Factor, Glutathione, and AIDS. Tumor Necrosis Factor: Structure-Function Relationship and Clinical Application. (3rd International Conference on Tumor Necrosis Factor and Related Cytokines, November 21-25, 1990, Makuhari, Chiba, Japan). T. Osawa and B. Bonavida. Basel, S. Karger, AG: 215-229.
Roederer, M., Staal, F. J., Osada, H. and Herzenberg, L. A. (1991). “CD4 and CD8 T cells with high intracellular glutathione levels are selectively lost as the HIV infection progresses.” International Immunology 3(9): 933-7.
Maintenance of intracellular glutathione (GSH) levels has been implicated in blocking cytokine-stimulated HIV replication in vitro, in both acute and latent infection models. We demonstrate here that subsets of human peripheral blood mononuclear cells differ substantially in mean GSH levels, as measured on a cell-by-cell basis with the fluorescence-activated cell sorter (FACS): B cells have the lowest GSH levels; T cells are intermediate; and monocytes and macrophages have the highest levels. Furthermore, GSH levels subdivide the CD4 and CD8 T cell subsets into two classes each: high- and low-GSH cells, which cannot be distinguished by cell size or by currently known surface markers. Significantly, the high-GSH T cells are selectively depleted early during the HIV infection, and are effectively missing in all ARC and AIDS patients.
Kerr, W. and Herzenberg, L. A. (1991). Gene-search viruses and FACs Fall permit the detection, isolation and characterization of mammalian cells with in situ fusions between cellular genes and E.coli lacZ. Methods: A Companion Volume to Methods in Enzymology, Academic Press. 2(3): 261-271.
Staal, F. J. T., Roederer, M., Israelski, D. M., Bubp, J., Mole, L. A., McShane, D., Deresinski, S. C., Ross, W., Sussman, H., Raju, P. A., Anderson, M. T., Moore, W., Ela, S. W., Herzenberg, L. A. and Herzenberg, L. A. (1992). “Intracellular glutathione levels in T cell subsets decrease in HIV infected individuals.” AIDS Res. Human Retrov. 8(2): 305-314.
The authors have shown previously that intracellular glutathione (GSH) plays an important role in the regulation of human immunodeficiency virus (HIV) transcription and replication in vitro, through modulation of signal transduction by inflammatory cytokines. Moreover, intracellular GSH levels are known to regulate T lymphocyte function. In multiparameter FACS studies presented here, we show that relative GSH levels in CD4+ and CD8+ T cells from HIV+ individuals are significantly lower than in corresponding subsets from uninfected controls. These studies define the relative intracellular glutathione (GSH) levels in CD4+ T cells, CD8+ T cells, B cells, and monocytes from 134 HIV infected individuals and 31 uninfected controls. The greatest decreases in intracellular GSH occur in subsets of T cells in individuals in the later stages of the HIV infection. In AIDS patients, GSH levels are 63 % of normal in CD4+ T cells (p < 0.0001) and are 62 % of normal in CD8+ T cells (p < 0.0001). Similarly, in AIDS-related complex (ARC) patients, GSH levels are 66% of normal in CD4+ T cells (p < 0.003) and are 69% of normal in CD8+ T cells (p < 0.003). These findings suggest that low intracellular GSH levels may be an important factor in HIV infection and in the resulting immunodeficiency.
Staal, F. J., Roederer, M., Bubp, J., Mole, L. A., Anderson, M. T., Raju, P. A., Israelski, D. M., Deresinski, S. C., Moore, W. A., Herzenberg, L. A. and et al. (1992). “CD20 expression is increased on B lymphocytes from HIV-infected individuals [published erratum appears in J Acquir Immune Defic Syndr 1992;5(9):956].” J Acquir Immune Defic Syndr 5(6): 627-32.
In studies presented here, we show that expression of the pan B cell marker CD20 is markedly increased on B lymphocytes from HIV-infected individuals and that this increase tends to be greater in individuals with more advanced disease. By using multiparameter FACS analyses to quantitate surface density of CD20 and intracellular glutathione (GSH) levels simultaneously, we further show that the distribution of intracellular glutathione (GSH) levels in B cells of HIV-infected individuals is more heterogeneous than in uninfected controls. Finally, we show that the intracellular GSH levels correlate with CD20 expression on a per-cell basis in all infected individuals. These findings suggest that CD20 expression, which can be precisely measured, may prove to be a useful surrogate marker for monitoring HIV infection.
Kantor, A. B., Stall, A. M., Adams, S. and Herzenberg, L. A. (1992). “Differential development of progenitor activity for three B-cell lineages.” Proceedings of the National Academy of Sciences of the United States of America 89(8): 3320-4.
Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-1 B cells (B-1a), which develop early and maintain their numbers by self-replenishment; and Ly-1B "sister" (B-1b) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which the immune response potential of each successive lineage is adapted to its particular niche.
Staal, F. J., Roederer, M. and Herzenberg, L. A. (1992). “Glutathione and immunophenotypes of T and B lymphocytes in HIV-infected individuals.” Annals of the New York Academy of Sciences 651: 453-63.
LAH #356-01
Herzenberg, L. A., Haughton, G. and Rajewsky, K., Eds. (1992). CD5 B Cells in Development and Disease. Annals of the N.Y. Acad. Sci. NY, N.Y. Acad. Sci.
Kantor, A. B. (1992). “CD5 B cells in development and disease.” Immunology Today.
Roederer, M., Ela, S. W., Staal, F. J. and Herzenberg, L. A. (1992). “N-acetylcysteine: a new approach to anti-HIV therapy.” AIDS Research & Human Retroviruses 8(2): 209-17.
Several investigators have implicated depletion of glutathione (GSH) and production of reactive oxygen intermediates (ROIs) in the regulation of the human immunodeficiency virus (HIV). We have shown directly that N-acetylcysteine (NAC) blocks HIV expression in chronic and acute infection models, and HIV replication in normal peripheral blood mononuclear cells. NAC is a cysteine prodrug which maintains intracellular thiol levels during oxidative stress and replenishes depleted GSH. The observed antiviral effect of NAC is due to inhibition of viral stimulation by ROIs, which are produced in response to inflammatory cytokines. We have also shown that HIV-infected individuals have decreased intracellular GSH levels in their circulating T cells. Since GSH is the major protection against the production of ROIs, we hypothesize that the observed decrease is due to a chronic oxidative stress induced by continual exposure to elevated levels of inflammatory cytokines. Together, these results provide a rationale for clinical trials testing the efficacy of GSH-replenishing drugs such as NAC in the treatment of AIDS. NAC is different than many other antiviral drugs in that it inhibits host-mediated stimulation of viral replication arising in normal immune responses, and may thereby extend latency. In addition, it inhibits the action of inflammatory cytokines which may mediate cachexia, thereby raising the possibility that it may alleviate the deleterious wasting that accompanies late stage AIDS.
Herzenberg, L. A. and Kantor, A. B. (1992). “Layered evolution in the immune system. A model for the ontogeny and development of multiple lymphocyte lineages.” Annals of the New York Academy of Sciences 651: 1-9.
Bhat, N. M., Kantor, A. B., Bieber, M. M., Stall, A. M., Herzenberg, L. A. and Teng, N. N. (1992). “The ontogeny and functional characteristics of human B-1 (CD5+ B) cells.” International Immunology 4(2): 243-52.
We demonstrate that, on average, greater than 90% of B lymphocytes in fetal spleen express CD5 at gestational ages of 17-23 weeks. Similarly, CD5+ B cells (B-1 cells) are the major B cell subset in umbilical cord blood. These findings depend on the optimization of fluorochrome conjugated anti-CD5 reagents for multiparameter fluorescent-activated cell sorter (FACS) analysis. From infancy through childhood the percentage of B-1 cells gradually diminishes in both spleen and peripheral blood. Stable adult levels, 25-35% of the total B cell population, are reached in late adolescence. The decrease in the percentage of B-1 cells in spleen is accompanied by an increase in conventional (CD5-) B cells, keeping the percentage of total B cells per mononuclear cells relatively constant. In contrast, in peripheral blood, the concentration of both B-1 cells and total B cells decreases, while T cells increase. At the functional level, we show that polyreactive IgM autoantibodies are produced by FACS-sorted CD5high B cells, but not by CD5- B cells from adolescent spleen. In contrast, fetal splenic CD5high and CD5- B cells appear functionally uniform, both producing IgM autoantibodies that are typical of B-1 cells. The apparent level of CD5- B cells in fetal spleen, on average 10% of total B cells, may still result from limitations of our reagent. The prominence of B-1 cells in fetal spleen and cord blood, the gradual reduction of B-1 cells with increasing age, and its characteristic repertoire, all suggest a role for this cell type in immunologically immature hosts.
Kroese, F. G. M., Kantor, A. B. and Herzenberg, L. A. (1994). The role of B-1 cells in mucosal immune responses. The role of B-1 cells in mucosal immune responses. P. L. M. D. Ogra, J. M. D. Mestecky, M. E. M. D. Lammet al. New York, Academic Press, Inc.: 217-222.
Kantor, A. B. (1991). “A new nomenclature for B cells.” Immunology Today 12: 388.
Kantor, A. B., Stall, A. M., Adams, S. and Herzenberg, L. A. (1992). “Adoptive transfer of murine B-cell lineages.” Annals of the New York Academy of Sciences 651: 168-9.
Stall, A. M., Adams, S., Herzenberg, L. A. and Kantor, A. B. (1992). “Characteristics and development of the murine B-1b (Ly-1 B sister) cell population.” Annals of the New York Academy of Sciences 651: 33-43.
In this paper we have outlined the evidence for two distinct branches of the B-1 cell lineage. The data show that phenotypically B-1a and B-1b cells are essentially identical, distinguished only by the presence or absence of the CD5 antigen. Functionally no differences between the two populations have yet been identified. Both produce anti-PtC antibodies, a specificity not observed in conventional B cells. Both produced high levels of IgM as measured in adoptive transfer experiments. Developmentally, B-1a and B-1b cells are indistinguishable with respect to generation from progenitors present in fetal liver and omentum, feedback regulation of new B-1a and B-1b cells from bone marrow, self-replenishment from Ig+ cells following adoptive transfer, and the generation of clonal populations. The major difference in the two populations is seen in the development of B-1a and B-1b cells from B220- progenitors in the adult bone marrow. Although B220- B-1a progenitors are rare in adult (greater than 6 weeks) bone marrow, the progenitors for B-1b cells persist well into adulthood. Our understanding of B-1b cell ontogeny is at a stage similar to that of B-1a cells five years ago. We have evidence from transfer experiments that strongly suggests the existence of two distinct progenitors for B-1a and B-1b, but we have yet to physically separate these progenitors as Solvansen et al. have done for B-1 and conventional B cells. Furthermore we must determine whether the B-1b cells that develop from fetal liver and bone marrow are functionally and developmentally equivalent to those that develop from adult bone marrow. As with B-1a cells, the role of B-1b cells in the immune system is unclear. Although we have not yet discerned functional differences between B-1a and B-1b, given the recent identification of CD72 (Lyb-2) as the ligand for CD5, it is tempting to speculate that B-1a cells are more involved in B-B cell interactions such as idiotype-anti-idiotype regulation of the early B-cell repertoire and that B-1b cells are more involved in B-T cell interactions. Whatever their function, it is clear that in trying to understand the role of the B-1 lineage it is important to consider both the B-1a and B-1b lineages.
Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T. and Herzenberg, L. A. (1992). “Glutathione deficiency and human immunodeficiency virus infection [see comments].” Lancet 339(8798): 909-12.
Roederer, M., Staal, F. J., Ela, S. W. and Herzenberg, L. A. (1993). “N-acetylcysteine: potential for AIDS therapy.” Pharmacology 46(3): 121-9.
The observations that people infected with HIV suffer not only from an inflammatory stress but also from depleted glutathione levels have led to a general hypothesis that these two are causally related, and that treatment of AIDS should include thiol-replenishment therapy. In particular, inflammatory stimulations are dependent on intracellular thiol levels, as they are potentiated at low glutathione levels (oxidative stress) and inhibited at high glutathione levels. Inflammatory stress may itself lead to decreased levels of glutathione. HIV has taken advantage of inflammatory signals to regulate its own replication; thus, the HIV infection is exacerbated by low levels of glutathione. We have shown that N-acetylcysteine can inhibit inflammatory stimulations, including that of HIV replication. Since N-acetylcysteine can replenish depleted glutathione levels in vivo, we suggest that it be used as an adjunct in the treatment of AIDS.
Kroese, F. G. M., Ammerlaan, Willem A.M., Kantor, Aaron B. (1993). “Evidence that intentinal IgA plasma cells in µ,k transgenic mice are derived from B-1 (Ly-1 B) cells.” International Immunology 5: 1317-1327.
Lalor, P., Bucci, C., Fornaro, M., Rattazzi, M. C., Nakauchi, H., Herzenberg, L. A. and Alberti, S. (1992). “Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.” Immunology 76(1): 95-102.
We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.
Staal, F. J., Roederer, M., Raju, P. A., Anderson, M. T., Ela, S. W. and Herzenberg, L. A. (1993). “Antioxidants inhibit stimulation of HIV transcription.” AIDS Research & Human Retroviruses 9(4): 299-306.
In studies presented here, we demonstrate that antioxidants regulate NF-kappa B activation and signal transduction pathways leading to HIV expression. We show (1) that N-acetyl-L-cysteine (NAC), an antioxidant and an efficient glutathione (GSH) precursor, inhibits NF-kappa B activation and HIV expression under conditions in which GSH is depleted and NAC cannot be converted to GSH, (2) that the D-stereoisomer of NAC and a wide variety of chemically unrelated antioxidants also inhibit NF-kappa B activation and/or transcription directed by the HIV LTR, and (3) that depletion of GSH, the principal intracellular antioxidant, augments HIV production in an acute infection model. Taken together, these findings suggest direct antioxidant action as the mechanism for inhibition of HIV transcription by NAC. They also confirm that GSH, acting in its capacity as an antioxidant, regulates HIV expression and that exogenous antioxidants can potentiate this regulation.
Lam, K. P., Herzenberg, L. A. and Stall, A. M. (1993). “A high frequency of hybridomas from M54 mu heavy chain transgenic mice initially co-express transgenic and rearranged endogenous mu genes.” International Immunology 5(9): 1011-22.
The M54 transgenic mouse line, which carries the 17.2.25 Ig mu heavy chain gene, rearranges Ig heavy chains and expresses both transgenic and endogenous mu. B cell lineage development is selectively impaired in these mice and cells that simultaneously express transgenic and endogenous mu ('double-producers') are common amongst the B cells and plasma cells that do develop. Weaver, Imanishi-kari, Baltimore and colleagues failed to obtain double-producing hybridomas from M54 mice; however, molecular and serologic studies presented here show that such hybridomas are readily generated. These hybridomas are extremely unstable and rapidly yield variants producing either transgenic or endogenous mu. Therefore the stable cloned lines we obtained, like Weaver et al., were almost all single or non-producers. We also found that the VH gene usage in our hybridomas was skewed towards the JH proximal (VHQ52, VH81X) families, supporting the idea that the expression of the M54 transgene alters the endogenous Ig repertoire.
Wells, S. M., Kantor, Aaron B., Stall, Alan M. (1994). “CD43 (S7) expression identified peripheral B cell subsets.” International Immunology 153: 5503-5515.
Kantor, A. B. and Herzenberg, L. A. (1993). “Origin of murine B cell lineages.” Annual Review of Immunology 11: 501-38.
Until recently, the hematopoietic stem cells (HSC) that appear early in ontogeny were thought to constitute a homogeneous, self-replenishing population whose developmental potential remains constant throughout the life of the animal. Studies reviewed here, however, demonstrated clear differences in the developmental potential of fetal and adult progenitor populations (including FACS-sorted HSC). These studies, which chart the ability of various progenitor sources to reconstitute functionally distinct B cell populations, define three B cell lineages: B-1a cells (CD5 B cells), derived from progenitors that are present in fetal omentum and fetal liver but are largely absent from adult bone marrow; B-1b cells ("sister" population), derived from progenitors that are present in fetal omentum, fetal liver, and also in adult bone marrow; and conventional B cells, whose progenitors are missing from fetal omentum but are found in fetal liver and adult bone marrow. B-1a and B-1b cells share many properties, including self-replenishment and feedback regulation of development. These B cell studies, in conjunction with evidence for a similar developmental switch for T cells and erythrocytes, suggest that evolution has created a "layered" immune system in which successive progenitors (HSC) reach predominance during development and give rise to differentiated cells (B, T, etc) responsible for progressively more complex immune functions.