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Roederer, M., Bigos, M., Nozaki, T., Stovel, R. T., Parks, D. R. and Herzenberg, L. A. (1995). “Heterogeneous calcium flux in peripheral T cell subsets revealed by five-color flow cytometry using log-ratio circuitry.” Cytometry 21(2): 187-96.
Calcium flux measurements of different subpopulations of cells by flow cytometry are important in understanding complex interactions in the immune system. This paper discusses the use of the difference of Log signals as a preferred method for obtaining this information simultaneously with other immunofluorescence parameters. We describe simple modifications to a commercial instrument that enables the measurement of calcium flux in addition to three immunofluorescence parameters. Finally, we show an application of this technique to measuring calcium flux of T cell subsets in human blood. We show that different subsets of peripheral CD4 T cells have significantly different capabilities to flux calcium after CD3 stimulation. These differences are related to the functional capacities of the cells within these subsets.
Katsikis, P. D., Wunderlich, E. S., Smith, C. A. and Herzenberg, L. A. (1995). “Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals.” Journal of Experimental Medicine 181(6): 2029-36.
Apoptosis (programmed cell death) of T lymphocytes has been proposed as a mechanism which plays an important role in the pathogenesis of human immunodeficiency virus (HIV) disease. Activation of Fas (CD95) can either result in costimulation of proliferation and cytokine production or in the induction of apoptosis of T lymphocytes. This raises the possibility that Fas is involved in the observed T cell apoptosis during HIV disease. In this report we show that peripheral blood CD4+ and CD8+ T lymphocytes from HIV-infected individuals undergo apoptosis in vitro in response to antibody stimulation (cross-linking) of Fas at a much higher frequency than from uninfected controls. This anti-Fas-induced T cell apoptosis is markedly higher than spontaneous T cell apoptosis in HIV-infected individuals. Antibodies against other members of the tumor necrosis factor (TNF)/nerve growth factor receptor family such as CD27, CD30, CD40, 4-1BB, p55 TNF receptor, p75 TNF receptor, and TNF receptor-related protein did not result in any increase of T cell apoptosis above that spontaneously observed in HIV+ individuals. Anti-Fas-induced apoptosis was much higher in symptomatic HIV-infected individuals; and the magnitude of anti-Fas-induced CD4+ T cell apoptosis correlated inversely with peripheral blood CD4+ T cell absolute counts. Surface expression of Fas on T cells was also found to be higher in HIV-infected individuals. Resting and activated CD4+ and CD8+ T cells both underwent apoptosis in response to anti-Fas antibody. L-Selectin positive memory CD4+ T cells were especially susceptible to anti-Fas-induced apoptosis. These findings show that CD4+ and CD8+ T lymphocytes in HIV-infected individuals are primed in vivo to undergo apoptosis in response to Fas stimulation, suggesting that Fas signaling may be responsible for the T lymphocyte functional defects and depletion observed in HIV disease.
Kantor, A. B., Merrill, C. E., MacKenzie, J. D., Herzenberg, L. A. and Hillson, J. L. (1995). “Development of the antibody repertoire as revealed by single-cell PCR of FACS-sorted B-cell subsets.” Annals of the New York Academy of Sciences 764: 224-7.
Roederer, M. and Herzenberg, L. A. (1996). “Changes in antigen densities on leukocyte subsets correlate with progression of HIV disease.” International Immunology 8(1): 1-11.
In a cross-sectional study of 154 HIV-infected and 33 uninfected healthy adults, we show that characteristic changes in the levels of expression of leukocyte surface antigens occur in the HIV-infected individuals. These changes, which collectively occur on virtually every leukocyte subset, are specific: a particular antigen may increase or decrease on one subset of PBMC but remain constant on another. Furthermore, within any particular subset, the levels of one or more antigens may change, while the levels of other surface antigens on the same cells remain constant. Some of these antigens density changes have been noted before, e.g. increased CD20 on B cells, and increased CD38 and HLA-DR on CD8 T cells. However, the multiparameter flow cytometry methodology used here reveals changes in a substantially larger number of surface markers, some of which are restricted to fine subsets of PBMC, such as naive or memory T cell subsets. For many of these antigens, the change in expression correlates with absolute CD4 counts >500/microl; others differ only in those with counts >100microl. The changes in antigen densities we observed on B and T cells are consistent with the observation of a persistent quasi-activated state of these cells in HIV-infected individuals. Similarly, the altered expression of the signal-transducing molecules CD7 and CD16 that we demonstrated for NK cells may correlate with the functional defects previously demonstrated in NK cells. Thus, measurements of antigen densities such as those demonstrated here may provide surrogate markers for the altered functional capacities of PBMC subsets in HIV-infected individuals, and may thereby provide a much simpler assay for immunocompetence than in vitro functional assays.
Tokuhisa, T., Herzenberg, Leonore A. (1996). Epitope-Specific Regulation of Antibody Responses. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science.
Kantor, A. B., Merrill, C.E.Merrill, J.L. Hillson (1996). Construction of cDNA from Single Unstimulated Mouse B Lymphocytes: Method and Application to the Study of Expressed Antibody Repertoires in FACS-Sorted Murine B Cell Subsets. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science: 13.1-13.6.
Gerstein, R. M. (1996). The Generation of Junctional Diversity by V(D)J Recombination. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science: 14.1-14.9.
Parks, D. R., Kantor, A.B., Roederer, M. (1996). Overview: Flow Cytometry and FACS. The Handbook of Experiemental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science: 46.1-46.2.
Kantor, A. B., M. Roederer (1996). FACS Analysis of Leukocytes. The handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science: 43.1-49.13.
Tung, J. W., Kantor, A. B. and L.A., H. (1997). Quantitative competitve PCR. The Handbook of Experimental Immunology. Boston, Balckwell Science. 2 Cell Surface and Messenger Molecules of the Immune System: 59.1-59.6.
Anderson, M. T., M., R., I., T., L.A., H. and L.A., H. (1997). Glutathione Measurement of the Flow Cytometer. Handbook of Experimental Immunology. W. Herzenberg, Blackwell, & Herzenberg. Boston, Blackwell Science. 2, Cell Surface and Messenger Molecules of the Immune System, Section of Flow Cytometry(FACS) Chapter 54: 54.1-54.9.
Katsikis, P. D., E.S. Wunderlich, M. Amano (1996). Apoptosis and the Immune System: Flow Cytometry Techniques for Measurement of Apoptosis. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science: 55.1-55.5.
Khan, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Muller, S., Kantor, A. B., Herzenberg, L. A. and et al. (1995). “Defective B cell development and function in Btk-deficient mice.” Immunity 3(3): 283-99.
Mutations in the Bruton's tyrosine kinase (Btk) gene have been linked to severe early B cell developmental blocks in human X-linked agammaglobulinemia (XLA), and to milder B cell activation deficiencies in murine X-linked immune deficiency (Xid). To elucidate unequivocally potential Btk functions in mice, we generated mutations in embryonic stem cells, which eliminated the ability to encode Btk pleckstrin homology or kinase domains, and assayed their effects by RAG2-deficient blastocyst complementation or introduction into the germline. Both mutations block expression of Btk protein and lead to reduced numbers of mature conventional B cells, severe B1 cell deficiency, serum IgM and IgG3 deficiency, and defective responses in vitro to various B cell activators and in vivo to immunization with thymus-independent type II antigens. These results prove that lack of Btk function results in an Xid phenotype and further suggest a differential requirement for Btk during the early stages of murine versus human B lymphocyte development.
Parks, D. R. (1996). Flow Cytometry Instrumentation and Measurements. The handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science.
Parks, D. R., M. Bigos (1996). Collection, Display and Analysis of Flow Cytometry Data. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell Science: 50.1-50.11.
Stall, A. M. (1996). Mouse Immunoglobulin Allotypes. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell: 27.1-27.16.
Lorincz, M., Roederer, M., Diwu, Z., Herzenberg, L. A. and Nolan, G. P. (1996). “Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase.” Cytometry 24(4): 321-9.
We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. Furthermore, both the beta-glucuronidase and the previously described E. coli beta-galactosidase enzymes have high specificities for their cognate substrates, allowing each reporter gene to be measured by FACS independently.
Murphy, D. B. and Herzenberg, L. A. (1997). Overview: Specialized Mouse Strains and Study of Gene Expression and Function. Handbook of Experimental Immunology 5th Edition Section 25, Chapter 149. W. Herzenberg, Blackwell, Herzenberg. Boston, Blackwell Sciences: 149.1-149.1.
Kantor, A. B., Merrill, C. E., Herzenberg, L. A. and Hillson, J. L. (1997). “An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells.” Journal of Immunology 158(3): 1175-86.
Previous studies conclude that the repertoire of B-1a (CD5+ B) cells is highly restricted. Studies here, which use FACS sorting and single-cell PCR methodology to develop an unbiased representation of the IgH repertoires of B-1a, B-1b, and conventional B cells from the peritoneal cavity, demonstrate that the B-1a cell repertoire is more diverse than previously thought. Furthermore, adult B-1a cells have significantly fewer noncoded nucleotide (N) insertions than conventional B cells. However, B-1a cells are not defined by the absence of these regions, since such insertions are present in two-thirds of B-1a cell transcripts. All three B cell populations use a wide spectrum of V(H), D, and J(H) elements and display considerable diversity in complementarity-determining region 3 (CDR3). However, characteristic differences in the repertoires of all three B cell populations also exist, suggesting different selective and/or developmental forces act to shape each repertoire.
Kerr, W. G., Heller, M. and Herzenberg, L. A. (1996). “Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains.” Proceedings of the National Academy of Sciences of the United States of America 93(9): 3947-52.
Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function.
Katsikis, P. D., Garcia-Ojeda, M. E., Wunderlich, E. S., Smith, C. A., Yagita, H., Okumura, K., Kayagaki, N., Alderson, M. and Herzenberg, L. A. (1996). “Activation-induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals.” International Immunology 8(8): 1311-7.
T cell apoptosis has been proposed as an important contributor to the functional defects and depletion of T cells in HIV-infected individuals. However, the mechanisms involved in this apoptosis have not been elucidated. We recently showed that peripheral blood T cells from HIV-infected individuals are especially susceptible to Fas antigen-induced apoptosis. In this study we examine the role of Fas, CTLA-4, tumor necrosis factor (TNF) receptors (TNFR) and CD30, receptors known to be involved in T cell activation-induced cell death (AICD), in the spontaneous and activation (anti-CD3)-induced apoptosis of peripheral blood T cells from asymptomatic HIV-infected individuals. We report here that spontaneous and activation-induced T cell apoptosis cannot be inhibited by reagents that block interactions of Fas, CTLA-4, p55 and p75 TNFR and CD30 with their respective ligands. We also show that IL-12, IFN-gamma, IL-4 and IL-10 cannot modify spontaneous, activation- and anti-Fas-induced apoptosis. Anti-Fas preferentially induced CD4+ T cell apoptosis whereas AICD induced apoptosis equally in CD4+ and CD8+ T cells. We conclude that T cell AICD in HIV infection is not mediated by Fas, thus indicating that Fas-induced and activation-induced T cell apoptosis are independent mechanisms of apoptosis which may play different roles in the pathogenesis of HIV infection.
Anderson, M. T., Tjioe, I. M., Lorincz, M. C., Parks, D. R., Herzenberg, L. A. and Nolan, G. P. (1996). “Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.” Proceedings of the National Academy of Sciences of the United States of America 93(16): 8508-11.
Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes. However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry. Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells. We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1. Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP. Each mutant retains only one of the two excitation peaks of the wild-type protein. GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines. Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.
Weirich, E., Rabin, R. L., Maldonado, Y., Benitz, W., Modler, S. and Herzenberg, L. A. (1998). “Neutrophil CD11b expression as a diagnostic marker for early-onset neonatal infection.” Journal of Pediatrics 132(3 Pt 1): 445-51.
OBJECTIVES: To determine whether neutrophil surface expression of CD11b predicts early-onset infection or suspected infection in at-risk infants. STUDY DESIGN: CD11b expression on peripheral blood neutrophils was determined by flow cytometry of whole blood samples. Blood (0.1 ml) was obtained from a convenience sample of at-risk infants admitted to the neonatal intensive care unit, stained with antibodies detecting CD11b and CD15, chilled, and analyzed within 8 hours. Blood for culture, blood counts, and C-reactive protein (CRP) determination was obtained simultaneously. Subjects were grouped on the basis of culture results and clinical signs, and investigators were blinded to CD11b level.
RESULTS: Of 106 subjects, seven had positive bacterial or viral cultures ("confirmed infection"), 17 had clinical signs of infection but negative cultures ("suspected infection"), and 82 had negative cultures and no clinical signs ("no infection"). Neutrophil CD11b was elevated in all infants with confirmed infection, 94% with suspected infection, and none with no infection. The negative and positive predictive values, sensitivity, and specificity were 100%, 99%, 96%, and 100%, respectively, for diagnosis of neonatal infection at initial evaluation. CD11b levels correlated with peak CRP (r2 = 0.76, p < 0.0001); however, CD11b was elevated at the time of admission in all five infants with proven bacterial infection, whereas CRP was normal until the second day in the neonatal intensive care unit in three of these five. Both infants with positive viral cultures had elevated CD11b, but the CRP levels remained within normal limits. The negative predictive value of neutrophil CD11b for identifying suspected or confirmed infection was 99%.
CONCLUSION: This assay for neutrophil CD11b is a promising test for exclusion of early-onset neonatal infection. If validated prospectively, this assay may reduce hospital and antibiotic use in the population of neonates at risk for early-onset infection.
Roederer, M., Kantor, A. B., Parks, D. R. and Herzenberg, L. A. (1996). “Cy7PE and Cy7APC: bright new probes for immunofluorescence.” Cytometry 24(3): 191-7.
We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a "brightness" (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a "brightness" comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC.
Roederer, M., Landay, A. (1996). Flow Cytometric Evaluation in AIDS. The Handbook of Experimental Immunology. L. Herzenberg, L. Herzenberg, C. Blackwell and D. Weir. Boston, Blackwell: 182.1-182.12.
Seidl, K. J., MacKenzie, J. D., Wang, D., Kantor, A. B., Kabat, E. A. and Herzenberg, L. A. (1997). “Frequent occurrence of identical heavy and light chain Ig rearrangements.” International Immunology 9(5): 689-702.
Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.
Herzenberg, L. A., De Rosa, S. C., Dubs, J. G., Roederer, M., Anderson, M. T., Ela, S. W. and Deresinski, S. C. (1997). “Glutathione deficiency is associated with impaired survival in HIV disease.” Proceedings of the National Academy of Sciences of the United States of America 94(5): 1967-72.
Glutathione (GSH), a cysteine-containing tripeptide, is essential for the viability and function of virtually all cells. In vitro studies showing that low GSH levels both promote HIV expression and impair T cell function suggested a link between GSH depletion and HIV disease progression. Clinical studies presented here directly demonstrate that low GSH levels predict poor survival in otherwise indistinguishable HIV-infected subjects. Specifically, we show that GSH deficiency in CD4 T cells from such subjects is associated with markedly decreased survival 2-3 years after baseline data collection (Kaplan-Meier and logistic regression analyses, P < 0.0001 for both analyses). This finding, supported by evidence demonstrating that oral administration of the GSH prodrug N-acetylcysteine replenishes GSH in these subjects and suggesting that N-acetylcysteine administration can improve their survival, establishes GSH deficiency as a key determinant of survival in HIV disease. Further, it argues strongly that the unnecessary or excessive use of acetaminophen, alcohol, or other drugs known to deplete GSH should be avoided by HIV-infected individuals.
Roederer, M., Parks D.R., Herzenberg L.A., Herzenberg L.A. (1996). Flow Cytometry. Encyclopedia of Immunology. R. a. Delves. London, Academic. 2nd Edition: 932-943.
Herzenberg, L. A., De Rosa S., Herzenberg L.A. (1998). Low Glutathione Levels in CD4 T Cells Predict Poor Survival in AIDS; N-Acetyclsteine May Improve Survival. Oxidative Stress in Cancer, AIDS and Neuodegerative Diseases. O. R. Monatagnier L, Pasquier C. Stanford, CA, Marcel Dekker, Inc. 1: 379-387.
Seidl, K. J., MacKenzie, J. D., Wang, D., Kantor, A. B., Kabat, E. A. and Herzenberg, L. A. (1997). “Recurrent identical rearrangement and repeated expression of identical heavy and light chains in single anti-phosphatidylcholine B cells.” Annals of the New York Academy of Sciences 815: 484-8.
LAH #430
de Waard, R., Dammers, P. M., Tung, J. W., Kantor, A. B., Wilshire, J. A., Bos, N. A., Herzenberg, L. A. and Kroese, F. G. (1998). “Presence of germline and full-length IgA RNA transcripts among peritoneal B-1 cells.” Developmental Immunology 6(1-2): 81-7.
Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline C alpha mRNA and mature C alpha mRNA transcripts. Germline C alpha and mature C alpha transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-1-cell population in CD5+ B-1a cells and CD5- B-1b cells, it was shown that in vivo expression of germline C alpha and mature C alpha transcripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.
Lorincz, M., Herzenberg, L. A., Diwu, Z., Barranger, J. A. and Kerr, W. G. (1997). “Detection and isolation of gene-corrected cells in Gaucher disease via a fluorescence-activated cell sorter assay for lysosomal glucocerebrosidase activity.” Blood 89(9): 3412-20.
Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene transfer therapy as a curative treatment for Gaucher disease. Limitations of this approach include the potential for insufficient engraftment of gene-corrected cells and incomplete transduction of hematopoietic stem cells using retroviral gene transfer. Overcoming these obstacles may be critical in the case of treatment for Gaucher disease type 1, because GC transduced cells have not been shown to have a growth advantage over noncorrected cells. Here, we describe the development and application of a novel, fluorescence-activated cell sorter based assay that directly quantitates GC activity at the single cell level. In a test of this application, fibroblasts from a Gaucher patient were transduced, and high expressing cells sorted based on GC activity. Reanalysis of cultured sorted fibroblasts reveals that these cells maintain high levels of enzymatic activity, compared with the heterogeneous population from which they were sorted. The assay is sufficiently sensitive to distinguish GC activity found in Gaucher patient monocytes from that in normal controls. Furthermore, preliminary results indicate that increased GC activity can be detected in transduced, CD34+ enriched peripheral blood mononuclear cells isolated from a Gaucher patient. This method should be a useful addition to current gene therapy protocols as a means to quantitatively assess gene correction of relevant cell populations and potentially purify transduced cells for transplantation.
Katsikis, P. D., Garcia-Ojeda, M. E., Torres-Roca, J. F., Greenwald, D. R. and Herzenberg, L. A. (1997). “HIV type 1 Tat protein enhances activation-but not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals.” International Immunology 9(6): 835-41.
T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5-tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.
Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A., Kerr, W. G. and Soriano, P. (1997). “Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells.” Proceedings of the National Academy of Sciences of the United States of America 94(8): 3789-94.
The ROSA beta geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral beta geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5' ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.
Amano, M., Baumgarth, N., Dick, M. D., Brossay, L., Kronenberg, M., Herzenberg, L. A. and Strober, S. (1998). “CD1 expression defines subsets of follicular and marginal zone B cells in the spleen: beta 2-microglobulin-dependent and independent forms.” Journal of Immunology 161(4): 1710-7.
We have used multicolor FACS analysis, immunohistology, and functional assays to study the expression of CD1 on B cell subsets from normal and beta 2m-/- mice. Two B cell subpopulations were identified that express high levels of CD1 in normal mice: splenic marginal zone B cells (IgMhigh IgDlow CD21high CD24intermediate CD23- CD43-) and a newly identified subpopulation of follicular B cells. The latter cells are unusual, because they are IgDhigh CD23+, like follicular B cells, but express high levels of CD21 and IgM, an expression pattern that is associated with marginal zone B cells. Therefore, the high-level expression of CD1 and CD21 was found to be closely associated on splenic B cells. Immunohistology confirmed the expression of CD1 on marginal zone B cells and on clusters of B cells in splenic follicles. Both the high-level CD1 expression by these cells and the low-level CD1 expression by subpopulations of B cells in the spleen, lymph node, peritoneal cavity, and bone marrow were markedly reduced in beta 2m-/- mice. Despite this, a CD1-restricted T cell clone proliferated vigorously in response to LPS-activated spleen cells that had been obtained from both beta 2m-/- and wild-type mice. This response was inhibited by the 3C11 anti-CD1 mAb. These results show the heterogeneity of B cell subsets in their expression of the beta 2m-dependent form of CD1. They further suggest that a beta 2m-independent form of CD1 is expressed on B cells that can stimulate T cells; however, this form is not easily visualized with the anti-CD1 mAb used here.
Roederer, M., DeRosa, S., Herzenberg, L. A. and Herzenberg, L. A. (1996). Immunology of T cells in AIDS: Dynamics revealed by 8-color flow cytometry. Handbook of Immunology. New Yokr, Marcel Deller, Inc.: 209-220.
Roederer, M., Raju, P. A., Mitra, D. K. and Herzenberg, L. A. (1997). “HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28.” Journal of Clinical Investigation 99(7): 1555-64.
In this report, we demonstrate that the T cell tropic strain of HIV, LAI, does not replicate in naive CD4 T cells stimulated by cross-linking CD3 and CD28. In contrast, LAI replicates well in memory CD4 T cells stimulated in the same way. Unlike this physiologically relevant stimulation, PHA stimulates productive LAI replication in both naive and memory T cells. These studies were conducted with highly purified (FACS-isolated) subsets of CD4 T cells identified by expression of both CD45RA and CD62L. Remixing of purified T cells showed that naive T cells do not suppress LAI replication in memory T cells and that memory T cells do not restore LAI expression in naive T cells. The suppression of productive LAI replication in naive T cells is not due to differential expression of viral coreceptors, nor is it due to inhibition of activation of the important HIV transcription factors, nuclear factor-kappaB and activator protein-1. The inherent resistance of naive T cells to productive HIV infection, coupled with their proliferative advantage as demonstrated here, provides a sound basis for proposed clinical therapies using ex vivo expansion and reinfusion of CD4 T cells from HIV-infected adults.
LAH #437
Roederer, M., Herzenberg L.A. (1997). Flow Cytometry: A Powerful Tool for Molecular Biology. The Encyclopedia of Molecular Biology 1st Edition. Creighton. New York, John Wiley and Sons: IN PRESS.
Watanabe, N., De Rosa, S. C., Cmelak, A., Hoppe, R., Herzenberg, L. A. and Roederer, M. (1997). “Long-term depletion of naive T cells in patients treated for Hodgkin's disease.” Blood 90(9): 3662-72.
We investigated the representation of T cells in patients who had been treated for Hodgkin's disease (HD). We found a marked depletion in both CD4 and CD8 naive T-cell counts that persists up to 30 years after completion of treatment. In contrast, CD4 and CD8 memory T-cell subsets recovered to normal or above normal levels by 5 years posttreatment. Thus, the previously-reported long-term deficit in total CD4 T-cell counts after treatment for HD is due to specific depletion of naive T cells. Similarly, total CD8 T-cell counts return to normal by 5 years only because CD8 memory T cells expand to higher than normal levels. These findings suggest that the treatment (mediastinal irradiation) results in a longterm dysregulation of T-cell subset homeostasis. The profound depletion of naive T cells may explain the altered T-cell function in treated patients, including the poor response to immunization after treatment for HD. Further, in some individuals, we identified expansions of unusual subsets expressing low levels of CD8. Eight-color fluorescence-activated cell sorting analyses showed that these cells largely express CD8alphaalpha homodimers and CD57, consistent with the phenotype of potentially extrathymically derived T cells. In addition, these cells, both CD4+ and CD4-, are probably cytotoxic lymphocytes, as they express high levels of intracellular perforin. In adults treated for HD, an increased activity of extrathymic T-cell differentiation may partially compensate for the loss of thymic-derived T cells.
Roederer, M., De Rosa, S., Gerstein, R., Anderson, M., Bigos, M., Stovel, R., Nozaki, T., Parks, D. and Herzenberg, L. (1997). “8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity.” Cytometry 29(4): 328-39.
We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems.
Roederer, M., De Rosa, S. C., Watanabe, N. and Herzenberg, L. A. (1997). “Dynamics of fine T-cell subsets during HIV disease and after thymic ablation by mediastinal irradiation.” Seminars in Immunology 9(6): 389-96.
The T-cell compartment is considerably more complex than just CD4 and CD8 T cells. Indeed, we can identify dozens of functionally and phenotypically distinct subsets within the peripheral blood of humans. These subsets are differentially affected in diseases which may underly some of the functional defects attributable to the disease. In HIV disease, all thymic-derived T-cell populations are gradually lost at identical rates during late-stage disease progression, while unusual, perhaps extrathymically-derived T cells expand. This expansion may reflect an attempt on the part of the immune system to compensate for the significant insult of HIV infection to the host: the abrogation of normal thymopoiesis and T-cell homeostasis. Copyright 1997 Academic Press Limited.
Katsikis, P. D., Garcia-Ojeda, M. E., Torres-Roca, J. F., Tijoe, I. M., Smith, C. A. and Herzenberg, L. A. (1997). “Interleukin-1 b converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection. TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection.” Journal of Experimental Medicine 186(8): 1365-72.
Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)-induced and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals. In this study, we extend and confirm these observations by using an inhibitor of interleukin-1 beta converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4(+) and CD8+ T cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)- induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this AICD is mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different roles in the pathogenesis of HIV infection.
Lorincz, M. C., Parente, M. K., Roederer, M., Nolan, G. P., Diwu, Z., Martin, D. I., Herzenberg, L. A. and Wolfe, J. H. (1999). “Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity.” Journal of Biological Chemistry 274(2): 657-65.
Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.
Anderson, M. T., Baumgarth, N., Haugland, R. P., Gerstein, R. M., Tjioe, T. and Herzenberg, L. A. (1998). “Pairs of violet-light-excited fluorochromes for flow cytometric analysis.” Cytometry 33(4): 435-44.
We describe pairs of fluorochromes for use with the 407-nm line of a violet-light-enhanced krypton ion laser. These fluorochromes and a previously described violet-light-excited reporter variant, GFP-Vex, fall into two emission classes: blue for Cascade Blue, and green/yellow for Cascade Yellow, Lucifer Yellow, and GFP-Vex. Cascade Yellow is a new fluorochrome that we have synthesized and is used for the first time in the present study. The two emission classes are sufficiently different that Cascade Blue can be paired with Cascade Yellow, Lucifer Yellow, or GFP-Vex in flow cytometric analysis. Furthermore, with proper detection filters, these fluorochromes can be combined with all of the currently used fluorochromes in a three-laser FACS system. With these data, the total number of fluorochromes that can be used as antibody labels for simultaneous detection in combined FACS analysis increases to nine. This study demonstrates the sensitivity and power of the combined use of these reagents in a single eight-color analysis by identifying murine T-lymphocyte subsets that could not otherwise be readily distinguished.
LAH #444
Torres-Roca, J., Greenwald, D., Brown, J., Weissman, I., Herzenberg, L., Herzenberg, L. and Katsikis, P. (1997). “Very-low oxygen conditions distinguish between oxygen-dependent and independent apoptosis.” J. of Allergy and Clin. Immun. 99(1/pt.2): 1276.
Galbraith, D. W., Anderson, M. T. and Herzenberg, L. A. (1999). “Flow cytometric analysis and FACS sorting of cells based on GFP accumulation.” Methods in Cell Biology 58: 315-41.
Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. and Herzenberg, L. A. (1999). “Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system.” Proceedings of the National Academy of Sciences of the United States of America 96(5): 2250-5.
"Natural" Igs, mainly IgM, comprise part of the innate immune system present in healthy individuals, including antigen-free mice. These Igs are thought to delay pathogenicity of infecting agents until antigen-induced high affinity Igs of all isotypes are produced. Previous studies suggested that the acquired humoral response arises directly from the innate response, i.e., that B cells expressing natural IgM, upon antigen encounter, differentiate to give rise both to cells that secrete high amounts of IgM and to cells that undergo affinity maturation and isotype switching. However, by using a murine model of influenza virus infection, we demonstrate here that the B cells that produce natural antiviral IgM neither increase their IgM production nor undergo isotype switching to IgG2a in response to the infection. These cells are distinct from the B cells that produce the antiviral response after encounter with the pathogen. Our data therefore demonstrate that the innate and the acquired humoral immunities to influenza virus are separate effector arms of the immune system and that antigen exposure per se is not sufficient to increase natural antibody production.
De Rosa, S. C., Zaretsky, M. D., Dubs, J. G., Roederer, M., Anderson, M., Green, A., Dipendra Mitra, M., Watanabe, N., Nakamura, H., Tjioe, I., Deresinski, S. C., Moore, W. A., Ela, S. W., Parks, D., Herzenberg, L. A. and Herzenberg, L. A. (2000). "N-acetylcysteine (NAC) replenishes glutathione in HIV infection." European Journal of Clinical Investigation: 30(10):915-929.
Background
Glutathione (GSH) deficiency is common in HIV-infected individuals and is associated
with impaired T cell function and impaired survival. N-acetylcysteine (NAC)
is used to replenish GSH that has been depleted by acetaminophen overdose. Studies
here test oral administration of NAC for safe and effective GSH replenishment
in HIV infection.
Design Oral NAC administration in a randomized, 8-week double-blind, placebo-controlled
trial followed by optional open-label drug for up to 24 weeks.
Subjects HIV-infected, low GSH, CD4 T cells < 500 mL -1 , no active opportunistic infections or other debilitation; h=81. Study conducted prior to introduction of protease inhibitors.
Results Whole blood GSH levels in NAC arm subjects significantly increased from 0.88 mM to 0.98 mM, bringing GSH levels in NAC-treated subjects to 89% of uninfected controls (P =0.03). Baseline GSH levels in the placebo group (0.91) remained essentially the same during the 8 week placebo-controlled trial. T cell GSH, adjusted for CD4 T cell count and b2-microglobulin levels, also increased in the NAC-treated subjects (P ‹0.04). Adverse effects were minimal and not significantly associated with NAC ingestion.
Conclusion. NAC treatment for 8 weeks safely replenishes whole blood GSH and T cell GSH in HIV-infected individuals. Thus, NAC offers useful adjunct therapy to increase protection against oxidative stress, improve immune system function and increase detoxification of acetaminophen and other drugs. These findings suggest that NAC therapy could be valuable in other clinical situations in which GSH deficiency or oxidative stress plays a role in disease pathology, e.g. rheumatoid arthritis, Parkinson's disease, hepatitis, liver cirrhosis, septic shock and diabetes.
Keywords glutathione, GSH, GSH deficiency, HIV, N-acetylcysteine, NAC
Seidl, K. J., Wilshire, J. A., MacKenzie, J. D., Kantor, A. B. and Herzenberg, L. A. (1999). “Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites.” Proceedings of the National Academy of Sciences of the United States of America 96(5): 2262-7.
Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.
Tung,
J. W., Kunnavatana, S. S. and Herzenberg, L. A. (2001). “The regulation of CD5
expression in murine T cells.” BMC Mol Biol 2(1): 5.
BACKGROUND: CD5 is a pan-T cell surface marker that is also present on
a subset of B cells, B-1a cells.Functional and developmental subsets of T cells
express characteristic CD5 levels that vary over roughly a 30- fold range. Previous
investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and
showed that it can confer similar lymphocyte-specific expression pattern as
observed for endogenous CD5 expression.
RESULTS: We further characterize the CD5 promoter and identify minimal
and regulatory regions on the CD5 promoter. Using a luciferase reporter system,
we show that a 43 bp region on the CD5 promoter regulates CD5 expression in
resting mouse thymoma EL4 T cells and that an Ets binding site within the 43
bp region mediates the CD5 expression. In addition, we show that Ets-1, a member
of the Ets family of transcription factors, recognizes the Ets binding site
in the electrophoretic mobility shift assay (EMSA). This Ets binding site is
directly responsible for the increase in reporter activity when co- transfected
with increasing amounts of Ets-1 expression plasmid.We also identify two additional
evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y) and demonstrate
the respective roles of the each region in the regulation of CD5 transcription.
CONCLUSION: Our studies define a minimal and regulatory promoter for
CD5 and show that the CD5 expression level in T cells is at least partially
dependent on the level of Ets-1 protein. Based on the findings in this report,
we propose a model of CD5 transcriptional
Anderson, M. T., Trudell, J. R., Voehringer, D. W., Tjioe, I. M. and Herzenberg, L. A. (1999). “An improved monobromobimane assay for glutathione utilizing tris- (2-carboxyethyl)phosphine as the reductant.” Analytical Biochemistry 272(1): 107-9.
Bigos, M., Baumgarth, N., Jager, G. C., Herman, O. C., Nozaki, T., Stovel, R. T., Parks, D. R. and Herzenberg, L. A. (1999). “Nine color eleven parameter immunophenotyping using three laser flow cytometry.” Cytometry 36(1): 36-45.
BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given.
METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals.
RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized.
CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.
Nakamura, H., De Rosa, S. C., Roedererd, M., Yodoi, J., Holmgren, A., Herzenberg, L. A. and Herzenberg, L. A. (2001). “Chronic elevation of plasma thioredoxin: inhibition of chemotaxis and curtailment of life expectancy in AIDS.” Proceedings of the National Academy of Sciences 98(5): 26885-2693.
Thioredoxin (Trx) is an intracellular redox protein with extracellular cytokine-like
and chemokine-like activities. We show here that although plasma TRX levels
do not influence the survival of HIV-infected individuals with CD4 cell counts
above 200/ul blood, survival is significantly impaired when plasma Trx is elevated
(p=0.003) in HIV-infected subjects with CD4 T cell counts below this level (i.e.,
with CDC-defined AIDS). In addition, we show that, as with other chemokines,
acute elevation of circulating Trx efficiently blocks LPS-induced chemotaxis
in mice. This block cripples innate immune mechanisms that are particularly
crucial when adaptive immunity is compromised, e.g., in the latter stages of
HIV disease. Thus, we propose that elevated plasma Trx directly impairs survival
by blocking pathogen-induced chemotaxis and hence effectively eliminating the
last (innate) barrier against establishment of opportunistic and other infections
in immunodeficient individuals.
Roederer, M., L.A. Herzenberg (1999). Flow Cytometry. Encyclopedia of Molecular Biology. Creighton. New York, NY, John Wiley & Sons, Inc.: 1-4.
Bertini, R., Howard, O. M., Dong, H. F., Oppenheim, J. J., Bizzarri, C., Sergi, R., Caselli, G., Pagliei, S., Romines, B., Wilshire, J. A., Mengozzi, M., Nakamura, H., Yodoi, J., Pekkari, K., Gurunath, R., Holmgren, A., Herzenberg, L. A. and Ghezzi, P. (1999). “Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells.” Journal of Experimental Medicine 189(11): 1783-9.
Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.
Voehringer,
D. W., Hirschberg, D. L., Xiao, J., Lu, Q., Roederer, M., Lock, C. B., Herzenberg,
L. A. and Steinman, L. (2000). “Gene microarray identification of redox and
mitochondrial elements that control resistance or sensitivity to apoptosis.”
Proc Natl Acad Sci U S A 97(6): 2680-2685.
Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.
Baumgarth, N., Jager, G. C., Herman, O. C. and Herzenberg, L. A. (2000). "CD4+ T cells derived from B cell-deficient mice inhibit the establishment of peripheral B cell pools." Proc Natl Acad Sci U S A 97(9): 4766-4771.
We demonstrate that adoptive transfer of peritoneal cavity B cells fails to replenish the peripheral B-1 cells in adult B cell-deficient (mu(-/-)) mice but does replenish adult RAG-1(-/-) mice. We show that this lack of self-replenishment in mu(-/-) mice is mediated by strongly inhibitory, radiation-sensitive CD4(+) T cells that also function in cotransfer studies to block the reconstitution of B-1 cells and inhibit accumulation of bone marrow-derived B-2 cells in the periphery in irradiated recipients. CD8(+) T cells from mu(-/-) do not mediate this inhibition. The inhibitory CD4(+) T cells develop early in life, because B-1 cell replenishment occurs normally when B-1 cells are transferred into mu(-/-) neonates. Thus, we conclude that the presence of B cells in the neonate conditions the CD4(+) T-cell population to permit the establishment and maintenance of normal B cell pools throughout life
LAH #460
Andrus, J. P., De Rosa, S. C., Tjioe, I. M., Frankel, L. R., Roederer, M., Herzenberg, L. A. and Herzenberg, L. A. (1999). “Depressed Lymphocyte glutathione levels in patients with septic shock.” Journal of Investigative Medicine 47(2 - Feb. 99): A155.
Galbraith, D. W., Herzenberg, L. A. and Anderson, M. T. (1999). Flow Cytometric Analysis of Transgene Expression in Higher Plants: Green Fluorescent Protein. Methods in Enzymology. Conn. New York, John Wiley and Sons. 302: 296-315.
LAH #462
Herzenberg, L. A. (1999). Introduction to the Second Edition. Flow Cytometry and Cell Sorting. Radbruch. Heidelberg, Springer-Verlag: i.
Lee, P. P., Yee, C., Savage, P. A., Fong, L., Brockstedt, D., Weber, J. S., Johnson, D., Swetter, S., Thompson, J., Greenberg, P. D., Roederer, M. and Davis, M. M. (1999). “Characterization of Circulating T cell specific for tumor-associated antigens in melanoma patients.” Nat. Med. 5(6): 677-685.
Mitra, D. K., De Rosa, S. C., Luke, A., Balamurugan, A., Khaitan, B. K., Tung, J., Mehra, N. K., Terr, A. I., O'Garra, A., Herzenberg, L. A. and Roederer, M. (1999). “Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases.” International Immunology 11(11): 1801-10.
We identified functionally polarized subsets of CD4 memory T cells on the basis of the expression of CD11a, CD45RA and CD62L. Within the several phenotypically distinct subsets of CD4 memory cells are two that, upon stimulation, produce primarily IL-4 (MT(2), CD45RA(-)CD62L(+)CD11a(dim)) or primarily IFN-gamma (MT(1), CD45RA(-)CD62L(-)CD11a(bright)). In addition, four other phenotypically distinct subsets of CD4 cells have unique cytokine profiles. To determine the clinical relevance of the representation of these cell types, we analyzed blood from patients with the chronic diseases leprosy and atopy. These diseases are characterized as immunologically polarized, since T cell responses in affected individuals are often strongly biased towards T(h)1 (dominated by IFN-gamma production) or T(h)2 (IL-4 production). We show here that this polarization reflects homeostatic or differentiation mechanisms affecting the representation of the functionally distinct subsets of memory CD4 T cells, MT(1) and MT(2). Significantly, the representation of the MT(1) and MT(2) subsets differs dramatically between subjects with tuberculoid leprosy (a T(h)1 disease), or lepromatous leprosy or atopic disease (T(h)2 diseases). However, there was no difference in the cytokine profiles of these or any of the other finely resolved CD4 subsets, when compared between individuals across all disease states. Thus, it is the representation of these subsets in peripheral blood that is diagnostic of the polarized state of the immune system.
Herzenberg, L. A., Moore, W. A. and De Rosa, S. C. (1999). “Estimation of missing values.” Lancet 354(9179): Correspondence.
Baumgarth, N., Herman, O. C., Jager, G. C., Brown, L. E., Herzenberg, L. A. and Chen, J. (2000). “B-1 and B-2 cell–derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection.” Journal of Experimental Medicine, IN PRESS JULY17, 192(2).
Baumgarth, N., Chen, J., Herman, O., Jager, G. and Herzenberg, L. (1999). The Role of B-1 and B-2 Cells in Immune Protection from Influenza Virus Infection. 16th Workshop on the Mechanisms of B Cell Neoplasia 1999, Lister Hill Auditorium, National Library of Medicine, NIH, Bethesda, MD, Springer-Verlag.
Baumgarth, N. and Roederer, M. (2000). "A practical approach to multicolor flow cytometry for immunophenotyping." J Immunol Methods 243(1-2): 77-97.
Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to simultaneously measure up to 11 distinct fluorescences and two scattered light parameters on each cell. Such advanced multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. They provide a large amount of novel information for each sample studied, an exquisitely accurate quantitation of even rare cell populations, and allow identification and characterization of novel cell subsets. In particular, this technology is proving crucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such identification and enumeration is important for understanding disease pathogenesis. However, multicolor flow cytometry comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful results. In this manuscript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and artefacts that may occur, and provide some suggestions for how to best overcome these hurdles. While inspired by the 11-color technology that we currently use, these principles apply to all flow cytometric experiments in which more than one fluorescent dye is used. http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?db=m&form=6&dopt=r&uid=0010986408.
Herzenberg, L., Baumgarth, N. and Wilshire, J. (1999). B-1 cell origins and VH repertoire determination. 16th Workshop on the Mechanisms of B Cell Neoplasia 1999, Lister Hill Auditorium, National Library of Medicine, NIH, Bethesda, MD, Springer-Verlag.
De Rosa, S., Herzenberg, L., Herzenberg, L. and Roederer, M. (2001). “11 color, 13 parameter Flow Cytometry: Identification of naive T cells by phenotype, function and T cell receptor.” Nature Medicine 7(2): 245-248.
Recent findings illustrate that diseases are often accompanied by changes in
the numbers or function of `fine' lymphocyte subsets, even if changes in the
bulk lymphocyte populations are not evident. In some cases, such changes may
provide powerful prognostic and diagnostic information.
LAH
#471
Ghezzi, P., B. Romines, M. Fratelli, I. Eberini, E. Gianazza, S. Casagrande,
T. Laragione, M. Mengozzi and L. A. Herzenberg (2002). "Protein glutathionylation:
coupling and uncoupling of glutathione to protein thiol groups in lymphocytes
under oxidative stress and HIV infection." Mol Immunol 38(10): 773-80.
We show here that exposure to oxidative stress induces glutathione (GSH) modification
of protein cysteinyl residues (glutathionylation) in T cell blasts. Treating
the cells with the oxidant diamide induces thiolation of a series of proteins
that can be detected by 2D electrophoresis when 35S-cysteine is used to label
the intracellular GSH pool. This thiolation is reversible, proteins are rapidly
dethiolated and GSH is released from proteins once the oxidants are washed and
the cells are allowed to recover. Dethiolation is dependent on the availability
of GSH and thiols, since it is inhibited by GSH-depleting agents and improved
by N-acetyl-L-cysteine (NAC). The capacity of these agents to reverse glutathionylation
is diminished in T cell blasts infected in vitro with HIV, which is known to
cause oxidative stress. Consistent with these findings, the activity of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), an enzyme known to be inhibited by glutathionylation,
is inhibited in diamide-treated cells and recovers rapidly when cells are allowed
to dethiolate. Further, GAPDH activity is diminished by GSH-depleting agents
and augmented by NAC. Thus, reversible glutathionylation of proteins can rapidly
shift the activity of a key metabolic enzyme and thereby result in dramatic,
reversible changes in cellular metabolism.
LAH
#472
Herzenberg, L. A., S. C. De Rosa and L. A. Herzenberg (2000). "Monoclonal
Antibodies and the FACS: complementary tools for immunobiology and medicine."
Immunology Today 21(8): 383-390.
The histories of monoclonal antibodies and FACS (flow cytometry instruments)
are as closely intertwined as their uses today in biology and medicine. This
"memoir" recounts the meeting and the mating of these two central
technologies, whose joint offspring now populate clinical and research laboratories
throughout the world.
The Fluorescence Activated Cell Sorter (FACS) was born in our laboratory, ca
1968, after a long but natural labor. As an infant, it was cantankerous and
slow and required intensive care and feeding. Nonetheless, it began to do productive
work quite rapidly and, by 1969, we announced its birth in a publication demonstrating
the automated separation of mammalian plasma cells as a function of intracellular
fluorescence3. Over the next two years, we introduced improvements, including
laser illumination (which replaced the earlier arc lamp) that made the nascent
FACS sensitive enough to detect fluorescent-labeled antibodies bound to mammalian
lymphoid cells. Thus, by 1971, we were able to count, sort and functionally
characterize T and B cells tagged with fluorescent antibodies or antigens that
bound to cell surface determinants4.
By 1972, we began to use the FACS to answer immunologically relevant questions by labeling cells with fluorochrome-coupled reagents (conventional antibodies and protein antigens) and following the fate of sorted cells in adoptive recipients. The first of these studies showed that antigen-binding cells are the precursors of antibody producing cells2. Next, in a series of B cell commitment studies5-9, we and our collaborators defined relationships between surface Ig isotype and subsequent isotype production and, in studies documenting allelic (haplotype) exclusion, showed that surface Ig allotype in allotype heterozygotes reveals the allotype production potential (chromosomal commitment for all isotypes) of B cells and their progeny. Collectively, results from these studies set the stage for today's understanding of the mechanisms of allelic exclusion, Ig chromosome rearrangement and overall B cell development.
The first FACS characterization of human T cells, B cells and other peripheral blood leukocytes were conducted during these early years10,11. In addition, substantial progress was made characterizing murine T cell surface markers and functions12-17. Thus, by the time commercial FACS instruments became available, the potential for using these instruments for immunological studies was well established.
LAH
#473
Herzenberg, L. A. (2000). "B-1 Cells: the lineage question revisisted."
Immunological Reviews 175: 9-21.
LAH
#474
Andrus, J. P., L. A. Herzenberg and S. C. DeRosa (2001). "Effects of legislation
restricting pack sizes of paracetamol on self poisoning. Paracetamol should
be packaged with its antidote." Bmj 323(7313): 634. Comment to the editor.
LAH
#475
Mengozzi, M., M. Malipatlolla, S. C. De Rosa, L. A. Herzenberg and M. Roederer
(2001). "Naive CD4 T cells inhibit CD28-costimulated R5 HIV replication
in memory CD4 T cells." Proc Natl Acad Sci U S A 98(20): 11644-9.
Stimulation with antibodies to CD3 and CD28 coimmobilized on beads can be used
to significantly expand T cells ex vivo. With CD4 T cells from HIV-infected
patients, this expansion usually is accompanied by complete suppression of viral
replication, presumed to be caused by down-regulation of the viral coreceptor
CCR5 and up-regulation of CCR5 ligands. Here we show that this suppression occurs
in total CD4 T cells acutely infected with R5 HIV, but not in purified CD62L(-)
memory CD4 T cells. The lack of complete suppression in these memory cells,
typically comprising 10-40% of total CD4 T cells, occurs despite high levels
of CCR5 ligand secretion and down-regulation of CCR5. Significantly, adding
back naive or CD62L(+) memory CD4 T cells inhibits the viral replication in
the CD62L(-) cells, with the naive cells capable of completely repressing the
virus. Although this inhibition was previously thought to be specific to bead-bound
anti-CD3/CD28 stimulation, we show that the same suppression is obtained with
sufficiently strong anti-CD3/B7.1 stimulation. Our results show that inhibitory
mechanisms, expressed predominantly by strongly stimulated naive CD4 T cells
and mediated independently of CCR5-binding chemokines, play a role in the inhibition
of R5 HIV replication in CD4 T cells upon CD28 costimulation.
LAH
#476
Perez, O. D., G. P. Nolan, D. Magda, R. A. Miller, L. A. Herzenberg and L. A.
Herzenberg (2002). "Motexafin gadolinium (Gd-Tex) selectively induces apoptosis
in HIV-1 infected CD4+ T helper cells." PNAS 99(4): 2270-2274.
Here, we show that motexafin gadolinium (Gd-Tex), a compound that promotes intracellular
oxidative stress, selectively induces apoptosis in HIV-1-infected CD4+ T cells
in IL-2-stimulated cultures of peripheral blood mononuclear cells infected in
vitro with HIV-1. This selective induction of apoptosis, which we detect by
FACS analysis of intracellular HIV/p24 and concomitant surface and apoptosis
marker expression, is abrogated by the glutathione precursor, N-acetyl-L-cysteine.
Importantly, it occurs at Gd-Tex concentrations that are not cytotoxic to uninfected
cells in the culture. These findings suggest that Gd-Tex may have therapeutic
utility as an anti-HIV agent capable of selectively targeting and removing HIV-infected
cells in an infected host.
LAH
#477
Tjioe, I., T. Legerton, J. Wegstein, L. A. Herzenberg and M. Roederer (2001).
"Phycoerythrin-allophycocyanin: A resonance energy transfer fluorochrome
for immunofluorescence." Cytometry 44(1): 24-9.
BACKGROUND: As immunofluorescence experiments become more complex, the demand
for new dyes with different properties increases. Fluorescent dyes with large
Stoke's shifts that are very bright and have low background binding to cells
are especially desirable. We report on the properties of the resonance energy
tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original
fluorescence resonance energy tandem dye described in the literature, but it
has not been utilized because of the difficulty of synthesizing and preparing
a consistent product.
METHODS: PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC. RESULTS: PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%.
CONCLUSION: PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.
Breithaupt, T. B., A. L. Shires, D. W. Voehringer and L. A. Herzenberg (2001). "Isoelectric focusing and enzyme overlay membrane analysis of caspase 3 activation." Anal Biochem 292(2): 313-6.
LAH
#480
Lu, L.-S., J. Tung, N. Baumgarth, O. Herman, M. Gleimer, L. A. Herzenberg and
L. A. Herzenberg (2002). "Identification of a germ-line pro-B cell subset
that distinguishes the fetal/neonatal from the adult B cell development pathway."
PNAS: 052715399.
Studies presented here show that the expression of CD4, MHC class II (Ia,) and
B220 cleanly resolves a major and a minor subset within the earliest pro-B cell
population (germ-line pro-B) in adult bone marrow (BM). The major subset expresses
intermediate B220 and low CD4 levels. The minor subset, which constitutes roughly
20% of the adult germ-line pro-B, expresses very low B220 levels and does not
express CD4. Ia is clearly detectable at low levels on the major germ-line pro-B
subset, both in wild-type adult mice and in gene-targeted mice (RAG2[-]/[-]
and {micro}MT), in which B cell development terminates before the pre-B cell
stage. A small proportion of cells in the more mature pro-B cell subsets (Hardy
Fractions B and C) also express Ia at this level. In contrast, Ia levels on
the minor subset are barely above (or equal to) background. Surprisingly, the
major germ-line pro-B cell subset found in adults is missing in fetal and neonatal
animals. All of the germ-line pro-B in these immature animals express a phenotype
(very low B220, no CD4, or Ia) similar to that of the minor pro-B cell subset
in adult BM. Because B cell development in fetal/neonatal animals principally
results in B-1 cells, these findings demonstrate that the B-1 development pathway
does not include the major germ-line pro-B subset found in adult BM and hence
identify a very early difference between the B-1 and -2 development pathways.
LAH
#481
Salomon, A. R., D. W. Voehringer, L. A. Herzenberg and C. Khosla (2000). "Understanding
and exploiting the mechanistic basis for selectivity of polyketide inhibitors
of F0F1-ATPase." Proc Natl Acad Sci U S A 97(26): 14766-14771.
Recently, a family of polyketide inhibitors of F(0)F(1)-ATPase, including apoptolidin,
ossamycin, and oligomycin, were shown to be among the top 0.1% most cell line
selective cytotoxic agents of 37,000 molecules tested