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LAH
#500
Herzenberg, L. A. and L. A. Herzenberg (2004). "Genetics, FACS, Immunology,
and Redox: A Tale of Two Lives Intertwined." Annual Review of Immunology
0(0).
We (Len and Lee Herzenberg) have worked separately and together for over fifty
years.This blending of independence and mutual reliance is reflected here as
we shift back and forth in telling the story of the laboratory we have led and
the life we have lived. The space provided for this manuscript is very generous.
Yet, calculated out, it amounts to roughly 100 words per year for each of us.
To make the most of this, we have written autobiography rather than history.
In many instances, we have referred only briefly, or not at all, to work that
had major influences on our thinking. In addition, we have adopted a policy
of naming the many students, fellows and collaborators with whom we have worked
only by referring to our joint work. We hope that the reader realizes that there
would be no biography worth writing were it not for the contributions made by
these and all of our other colleagues. Expected online publication date for
the Annual Review of Immunology Volume 22 is March 19, 2004. Please see http://www.annualreviews.org/catalog/pub_dates.asp
for revised estimates.
LAH #501-1 Supporting Material
LAH
#501-2 Supporting Material
Yang, Y., C. H. Contag, et al. (2004). "The E47 transcription factor negatively
regulates CD5 expression during thymocyte development." Proc Natl Acad
Sci U S A 101(11): 3898-902.
The expression of CD5 increases progressively as thymocytes mature. We have
shown that CD5 expression is controlled by a tissue-specific regulatory promoter
located upstream of the CD5 translation start sites. Deletion of this regulatory
promoter, which contains three potential transcription factor binding sites
(CCAAT, kappa E2, and ets) reduces the promoter activity to basal level. Of
these sites, only ets proved essential for CD5 expression in T cell lines. Here,
we introduce a role for the E47 transcription factor and the CD5 promoter kappa
E2 site in regulating CD5 expression during thymocyte development. Using T cell
lines, we show that (i) mutation of the kappa E2 site in the CD5 regulatory
promoter results in a significant elevation of CD5 promoter activity; (ii) the
E47 transcription factor binds to the kappa E2 site; and (iii) overexpression
of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated
cell sorting studies with primary thymocytes at successive developmental stages,
that (i) intracellular E47 levels decrease as surface CD5 expression increases;
(ii) E47 expression is down-regulated and CD5 expression is correspondingly
up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3
to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated
and CD5 expression is up-regulated when double positive thymocytes are stimulated
in vitro with anti-CD3. Based on these data, we propose that E47 negatively
regulates CD5 expression by interacting with the kappa E2 site in the CD5 regulatory
promoter and that decreases in E47 in response to developmental signals are
critical to the progressive increase in CD5 expression as thymocytes mature.
LAH
#502-1 Related Material
Herzenberg, L. A. (2004). "FACS innovation: a view from Stanford."
Clin Invest Med 27(5): 240-52.
LAH
#503
Davidson, C., R. Tirouvanziam, L. Herzenberg and J. Lipsick (2004). "Functional
Evolution of the Vertebrate Myb Gene Family: B-Myb, but neither A-Myb nor c-Myb,
complements Drosophila Myb in Hemocytes." Genetics.
The duplication of genes and genomes is believed to be a major force in the
evolution of eukaryotic organisms. However, different models have been presented
about how duplicated genes are preserved from elimination by purifying selection.
Preservation of one of the gene copies due to rare mutational events that result
in a new gene function (neo-functionalization) necessitates that the other gene
copy retain its ancestral function. Alternatively, preservation of both gene
copies due to rapid divergence of coding and non-coding regions such that neither
retains the complete function of the ancestral gene (sub-functionalization)
may result in a requirement for both gene copies for organismal survival. The
duplication and divergence of the tandemly arrayed homeotic clusters have been
studied in considerable detail and have provided evidence in support of the
sub-functionalization model. However, the vast majority of duplicated genes
are not clustered tandemly, but instead are dispersed in syntenic regions on
different chromosomes, most likely as a result of genome-wide duplications and
rearrangements. The Myb oncogene family provides an interesting opportunity
to study a dispersed multigene family because invertebrates possess a single
Myb gene, whereas all vertebrate genomes examined thus far contain three different
Myb genes (A-Myb, B-Myb and c-Myb). A-Myb and c-Myb appear to have arisen by
a second round of gene duplication, which was preceded by the acquisition of
a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated
from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears
to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific
requirements during spermatogenesis and hematopoiesis, respectively. We now
report that the absence of Drosophila Myb (Dm-Myb) causes a failure of larval
hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes
from mosaic larvae reveal a phagocytosis defect. In addition, we show that vertebrate
B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte
proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause
lethality in the presence or absence of endogenous Dm-Myb. These results are
consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a
duplicated B-Myb-like gene. In addition, our results suggest that B-Myb and
Dm-Myb share essential conserved functions that are required for cell proliferation.
Finally, these experiments demonstrate the utility of genetic complementation
in Drosophila to explore the functional evolution of duplicated genes in vertebrates.
LAH
#504
Cao, Y. A., M. H. Bachmann, et al. (2005). "Molecular imaging using labeled
donor tissues reveals patterns of engraftment, rejection, and survival in transplantation."
Transplantation 80(1): 134-9.
Tissue regeneration and transplantation of solid organs involve complex processes
that can only be studied in the context of the living organism, and methods
of analyzing these processes in vivo are essential for development of effective
transplantation and regeneration procedures. We utilized in vivo bioluminescence
imaging (BLI) to noninvasively visualize engraftment, survival, and rejection
of transplanted tissues from a transgenic donor mouse that constitutively expresses
luciferase. Dynamic early events of hematopoietic reconstitution were accessible
and engraftment from as few as 200 transplanted whole bone marrow (BM) cells
resulted in bioluminescent foci in lethally irradiated, syngeneic recipients.
The transplantation of autologous pancreatic Langerhans islets and of allogeneic
heart revealed the tempo of transplant degeneration or immune rejection over
time. This imaging approach is sensitive and reproducible, permits study of
the dynamic range of the entire process of transplantation, and will greatly
enhance studies across various disciplines involving transplantation.
LAH #505
Gernez, Y., Herzenberg, L. A., Herzenberg, L. A. and Tirouvanziam, R. (2007). "[Phospho-Facs: A Powerful Tool for Exploring Intracellular Transduction Cascades.]." Rev Mal Respir 24(8): 955-64.
INTRODUCTION: FACS (fluorescence-activated cell sorting), or flow cytometry, was developed in 1971 by Leonard Herzenberg's team at Stanford University. Under continuous development, this technology enables single-cell multiparametric analysis and sorting, based on physical properties of cells and/or their relative expression levels of specific glycoproteic epitopes and metabolites.
STATE OF THE ART: Recently, the use of fluorescent antibodies specific for phosphorylated epitopes - or "phospho-epitopes" - within proteins of interest has further extended the range of FACS analyses. This new application, dubbed "phospho-FACS", has quickly become a tool of choice for delineating intracellular phosphorylation cascades.
PERSPECTIVES: In both basic research and clinical research, the application of phospho-FACS to cellular subsets from blood or the periphery, whether frequent or rare, enables the discovery of pathological biomarkers and therapeutic innovation.CONCLUSIONS: Thanks to its rapid implementation and its ability to generate single-cell data, the phospho-FACS technique features numerous advantages compared to preexisting analytical methods for intracellular phosphorylation cascades
LAH
#506
Baumgarth, N., J. W. Tung and L. A. Herzenberg (2005). "Inherent specificities
in natural antibodies: a key to immune defense against pathogen invasion."
Springer Semin Immunopathol.
Natural antibodies are produced at tightly regulated levels in the complete
absence of external antigenic stimulation. They provide immediate, early and
broad protection against pathogens, making them a crucial non-redundant component
of the humoral immune system. These antibodies are produced mainly, if not exclusively,
by a subset of long-lived, self-replenishing B cells termed B-1 cells. We argue
here that the unique developmental pattern of these B-1 cells, which rests on
positive selection by self antigens, ensures production of natural antibodies
expressing evolutionarily important specificities that are required for the
initial defense against invading pathogens. Positive selection for reactivity
with self antigens could also result in the production of detrimental anti-self
antibodies. However, B-1 cells have evolved a unique response pattern that minimizes
the risk of autoimmunity. Although these cells respond rapidly and strongly
to host-derived innate signals, such as cytokines, and to pathogen-encoded signals,
such as lipopolysaccharide and phosphorylcholine, they respond very poorly to
receptor-mediated activation. In addition, they rarely enter germinal centers
and undergo affinity maturation. Thus, their potential for producing high-affinity
antibodies with harmful anti-self specificity is highly restricted. The positive
selection of B-1 cells occurs during the neonatal period, during which the long-lived
self-renewing B-1 population is constituted. Many of these cells (B-1a) express
CD5, although a smaller subset (B-1b) does not express this surface marker.
Importantly, B-1a cells should not be confused with short-lived anergic B-2
cells, which originate in the bone marrow in adults and initiate CD5 expression
and programmed cell death following self-antigen recognition. In summary, we
argue here that the mechanisms that enable natural antibody production by B-1
cells reflect the humoral immune system, which has evolved in layers whose distinct
developmental mechanisms generate complementary repertoires that collectively
operate to maximize flexibility in responses to invading pathogens. B-2 cells,
present in what may be the most highly evolved layer(s), express a repertoire
that is explicitly selected against self recognition and directed towards the
generation of high-affinity antibody response to external antigenic stimuli.
B-1 cells, whose repertoire is selected by recognition of self antigen, belong
to what may be earlier layer(s) and inherently maintain production of evolutionarily
important antibody specificities that respond to pathogen-related, rather then
antigen-specific signals.
LAH
#507
Atkuri, K. R., L. A. Herzenberg and L. A. Herzenberg (2005). "Culturing
at atmospheric oxygen levels impacts lymphocyte function." Proc Natl Acad
Sci U S A 102(10): 3756-9.
To determine whether culturing peripheral blood mononuclear cells at atmospheric
oxygen levels skews responses in comparison with culturing lymphocytes at physiologic
oxygen levels, we cultured peripheral blood mononuclear cells at 5%, 10%, and
atmospheric (20%) gas-phase oxygen for 5 days. We found that incubator oxygen
levels influenced lymphocyte proliferation stimulated by two commonly used stimuli:
Con A and antibodies that crosslink surface CD3 and CD28 to mimic antigen presentation.
In both cases, proliferation increased as gas-phase oxygen levels increased.
In contrast, oxygen levels did not influence proliferation stimulated by phytohemagglutinin,
another commonly used mitogen. Similarly, oxygen levels did not impact cell
viability in unstimulated cultures. Thus, we conclude that the influence of
oxygen levels on proliferation depends on the stimulus, and, most importantly
from the standpoint of immune responses, culturing cells at atmospheric rather
than physiologic oxygen levels results in significantly increased proliferation
responses to the CD3/CD28 crosslinking, a proliferation stimulus commonly used
to mimic T cell antigen receptor signaling
LAH
#508
Sahaf, B., K. Heydari, L. A. Herzenberg and H. L. A. (2005). "The extracellular
microenvironment plays a key role in regulating the redox status of cell surface
proteins in HIV-infected subjects." Archives of Biochemistry and Biophysics
434(1): 26-32.
There is an overwhelming interest in the study of the redox status of the cell
surface affecting redox signaling in the cells and also predicting the total
redox status of the cells. Measuring the total surface thiols (cell surface
molecule thiols, csm-SH) we have shown that the overall level of surface thiols
is tightly controlled. In vitro, the total concentration of intracellular glutathione
(iGSH) seems to play a regulatory role in determination of the amounts of reduced
proteins on cells. In addition, short term exposure of the cell surface to glutathione
disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH
suggesting that the function of some cysteine containing proteins on the cell
surface may be regulated by the amount of GSSG secreted from the cells or the
GSSG available in the extracellular environment. Examination of peripheral blood
mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal
a similar correlation between the intra- and extracellular thiol status of cells.
Although there is a relatively wide variation between individuals in both csm-SH
and iGSH there is no correlation between the iGSH and csm-SH levels measured
for healthy and HIV-infected individuals. There are many reports suggesting
different redox active proteins on the cell surface to be the key players in
the total cell surface redox regulation. However, we suggest that the redox
status of the cells is regulated through a complex and tightly regulated mechanism
that needs further investigation. In the mean time, overall surface thiol measurements
together with case specific protein determinations may offer the most informative
approach. In this review, we discuss our own results as well as results from
other laboratories to argue that the overall levels of surface thiols on the
exofacial membrane are regulated primarily by redox status of the cell surface
microenvironment.
LAH #509
Gernez, Y., Tirouvanziam, R., Nguyen, K. D., Herzenberg, L. A., Krensky, A. M. and Nadeau, K. C. (2007). "Altered Phosphorylated Signal Transducer and Activator of Transcription Profile of Cd4(+)Cd161(+) T Cells in Asthma: Modulation by Allergic Status and Oral Corticosteroids." J Allergy Clin Immunol.
BACKGROUND: Asthma is a complex immunologic disorder linked to altered cytokine signaling.
OBJECTIVE: We tested whether asthmatic patients showed any change in cytokine-dependent signal transducer and activator of transcription (STAT) levels, focusing on the central/effector-memory CD4(+)CD161(+) subset, which represents 15% to 25% of circulating T cells. METHODS: We quantified intracellular levels of active phosphorylated STAT (phospho-STAT) 1, 3, 5, and 6 by means of flow cytometry, without any activation or expansion. RESULTS: Baseline phospho-STAT1 and phospho-STAT6 levels were increased in CD4(+)CD161(+) T cells from asthmatic patients compared with those from healthy control subjects (by 10- and 8-fold, respectively). This asthma-associated alteration was both subset specific because no change was seen in CD4(+)CD161(-)CD25(+) (regulatory T cells) and CD4(+)CD161(-)CD25(-) subsets and isoform specific because phospho-STAT5 and phospho-STAT3 levels were unchanged. Among asthmatic patients, phospho-STAT1 and phospho-STAT6 levels correlated negatively with each other, suggesting antagonistic regulation. Oral corticosteroid (OCS) treatment significantly decreased phospho-STAT6 and IL-4 levels but not phospho-STAT1 levels. Disease parameters showing significant correlations with phospho-STAT1, phospho-STAT6, or both included age at onset, plasma IgE levels, and levels of the T(H)2 cytokines IL-4 and IL-10 and the T(H)1 cytokine IL-2. Overall, combined phospho-STAT1 and phospho-STAT6 measurements showed excellent predictive value for identifying (1) asthmatic patients versus healthy control subjects, (2) allergic versus nonallergic asthmatic patients, and (3) asthmatic patients taking versus those not taking OCSs.
CONCLUSION: Baseline changes in phospho-STAT1 and phospho-STAT6 levels in blood CD4(+)CD161(+) T cells identify asthmatic patients and mirror their allergic status and response to OCSs.
CLINICAL IMPLICATIONS: These results confirm the pathologic importance of activated STAT1 and STAT6 in asthma and suggest their potential use as clinical biomarkers.
LAH
#510
Ray, S., K. R. Atkuri, D. Deb-Basu, A. S. Adler, H. Y. Chang, L. A. Herzenberg and D. W. Felsher (2006). "MYC Can Induce DNA Breaks In vivo and In vitro Independent of Reactive Oxygen Species." Cancer Res 66(13): 6598-6605.
MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production. (Cancer Res 2006; 66(13): 6598-605).
MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production. (Cancer Res 2006; 66(13): 6598-605).
LAH #511
Sanyal, M., Tung, J. W., Karsunky, H., Zeng, H., Selleri, L., Weissman, I. L., Herzenberg, L. A. and Cleary, M. L. (2007). "B Cell Development Fails in the Absence of the Pbx1 Proto-Oncogene." Blood.
Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B cell development from CLP following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19(+)) cells and thereafter, revealed that Pbx1 is not necessary for B cell development to proceed from the pro-B cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B cell commitment, and therefore is one of the earliest-acting transcription factors that regulate de novo B lineage lymphopoiesi
LAH
#512
Tung, J. W., M. D. Mrazek, Y. Yang, L. A. Herzenberg and L. A. Herzenberg (2006). "Phenotypically distinct B cell development pathways map to the three B cell lineages in the mouse." Proc Natl Acad Sci U S A 103(16): 6293-8.
A recent article by Montecino-Rodriguez et al. [Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. (2006) Nat. Immunol.7, 293-301] has distinguished the early progenitors for B-1 cells, which principally develop in neonates, from early progenitors for B-2 cells, which principally develop in adult bone marrow. Here we introduce syndecan-1 (CD138) and MHC class II (I-A) as markers of early B cell development [Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. & Hayakawa, K. (1991) J. Exp. Med. 173, 1213-1225; Hardy fractions B-D] and show that the expression of these markers distinguishes the predominant B cell development pathway in neonates from the corresponding predominant pathway in adults (both progenitors are present but differently represented in each case). We show that pre-B cells (Hardy fraction D) in the predominant adult pathway express high levels of CD138 and intermediate levels of I-A, whereas the corresponding pre-B cells in the pathway that predominates in neonates do not express either of these markers. As expected, because most of the pre-B cells in adults express CD138, we find that sorted CD138+ adult pre-B cells differentiate to IgM+ B cells in vitro. Sorted CD138- pre-B cells from neonates, the majority subset at this age, also mature to IgM+ cells (without passing through a CD138+ stage). Importantly, our studies here confirm the differential representation of adult and neonatal progenitor populations and further demonstrate that CD138 expression subdivides the adult CD19+, B220-6B2-/low population shown to contain B-1 progenitors in a way consistent with the predominance of B-1b progenitors in adults. Thus, CD138 expression provides a key route to distinguishing early B cell development pathway for what now are clearly three B cell lineages.
LAH
#513
Tirouvanziam, R., C. K. Conrad, T. Bottiglieri, L. A. Herzenberg, R. B. Moss
and L. A. Herzenberg (2006). "High-dose oral N-acetylcysteine, a glutathione
prodrug, modulates inflammation in cystic fibrosis." Proc Natl Acad Sci
U S A 103(12): 4628-33.
Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high
oxidant producers, airway neutrophils contribute largely to the systemic redox
imbalance seen in CF. In turn, this chronic and profound imbalance can impact
circulating neutrophils before their migration into airways. Indeed, in 18 CF
patients with stable disease, blood neutrophils were readily deficient in the
pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls).
In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione
prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times
daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase
activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently,
neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as
was the number of airway neutrophils actively releasing elastase-rich granules
(P = 0.005), as measured by flow cytometry. Pulmonary function measures were
not improved, as expected with short-term treatment. After excluding data from
subjects without baseline airway inflammation, positive treatment effects were
more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus,
high-dose oral N-acetylcysteine has the potential to counter the intertwined
redox and inflammatory imbalances in CF.
Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF). As high
oxidant producers, airway neutrophils contribute largely to the systemic redox
imbalance seen in CF. In turn, this chronic and profound imbalance can impact
circulating neutrophils before their migration into airways. Indeed, in 18 CF
patients with stable disease, blood neutrophils were readily deficient in the
pivotal antioxidant glutathione (P = 0.003, compared with 9 healthy controls).
In a phase 1 study, this deficiency was improved (P = 0.025) by the glutathione
prodrug N-acetylcysteine, given orally in high doses (0.6 to 1.0 g three times
daily, for 4 weeks). This treatment was safe and markedly decreased sputum elastase
activity (P = 0.006), the strongest predictor of CF pulmonary function. Consistently,
neutrophil burden in CF airways was decreased upon treatment (P = 0.003), as
was the number of airway neutrophils actively releasing elastase-rich granules
(P = 0.005), as measured by flow cytometry. Pulmonary function measures were
not improved, as expected with short-term treatment. After excluding data from
subjects without baseline airway inflammation, positive treatment effects were
more pronounced and included decreased sputum IL-8 levels (P = 0.032). Thus,
high-dose oral N-acetylcysteine has the potential to counter the intertwined
redox and inflammatory imbalances in CF.
LAH
#514
Weerkamp, F., M. R. Baert, B. A. Naber, E. E. Koster, E. F. de Haas, K. R. Atkuri,
J. J. van Dongen, L. A. Herzenberg and F. J. Staal (2006). "Wnt signaling
in the thymus is regulated by differential expression of intracellular signaling
molecules." Proc Natl Acad Sci U S A 103(9): 3322-6.
Wnt signaling is essential for T cell development in the thymus, but the stages
in which it occurs and the molecular mechanisms underlying Wnt responsiveness
have remained elusive. Here we examined Wnt signaling activity in both human
and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter
activation and expression of Wnt-target genes. We demonstrate that Wnt signaling
occurs in all thymocyte subsets, including the more mature populations, but
most prominently in the double negative (DN) subsets. This differential sensitivity
to Wnt signaling was not caused by differences in the presence of Wnts or Wnt
receptors, as these appeared to be expressed at comparable levels in all thymocyte
subsets. Rather, it can be explained by high expression of activating signaling
molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1,
and by low levels of inhibitory molecules. By blocking Wnt signaling from the
earliest stage onwards using overexpression of Dickkopf, we show that inhibition
of the canonical Wnt pathway blocks development at the most immature DN1 stage.
Thus, responsiveness to developmental signals can be regulated by differential
expression of intracellular mediators rather than by abundance of receptors
or ligands.
Wnt signaling is essential for T cell development in the thymus, but the stages
in which it occurs and the molecular mechanisms underlying Wnt responsiveness
have remained elusive. Here we examined Wnt signaling activity in both human
and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter
activation and expression of Wnt-target genes. We demonstrate that Wnt signaling
occurs in all thymocyte subsets, including the more mature populations, but
most prominently in the double negative (DN) subsets. This differential sensitivity
to Wnt signaling was not caused by differences in the presence of Wnts or Wnt
receptors, as these appeared to be expressed at comparable levels in all thymocyte
subsets. Rather, it can be explained by high expression of activating signaling
molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1,
and by low levels of inhibitory molecules. By blocking Wnt signaling from the
earliest stage onwards using overexpression of Dickkopf, we show that inhibition
of the canonical Wnt pathway blocks development at the most immature DN1 stage.
Thus, responsiveness to developmental signals can be regulated by differential
expression of intracellular mediators rather than by abundance of receptors
or ligands.
LAH #515
Herzenberg, L. A. and J. W. Tung (2006). "B cell lineages: documented at
last!" Nat Immunol 7(3): 225-6.
News and views.
LAH #516-1 Supplemental Material
Herzenberg, L. A., J. Tung, W. A. Moore, L. A. Herzenberg and D. R. Parks (2006). "Interpreting flow cytometry data: a guide for the perplexed." Nat Immunol 7(7): 681-5.
LAH
#517
Parks, D. R., M. Roederer and W. A. Moore (2006). "A new "Logicle" display method avoids deceptive effects of logarithmic scaling for low signals and compensated data." Cytometry A 69(6): 541-51.
BACKGROUND: In immunofluorescence measurements and most other flow cytometry applications, fluorescence signals of interest can range down to essentially zero. After fluorescence compensation, some cell populations will have low means and include events with negative data values. Logarithmic presentation has been very useful in providing informative displays of wide-ranging flow cytometry data, but it fails to adequately display cell populations with low means and high variances and, in particular, offers no way to include negative data values. This has led to a great deal of difficulty in interpreting and understanding flow cytometry data, has often resulted in incorrect delineation of cell populations, and has led many people to question the correctness of compensation computations that were, in fact, correct. RESULTS: We identified a set of criteria for creating data visualization methods that accommodate the scaling difficulties presented by flow cytometry data. On the basis of these, we developed a new data visualization method that provides important advantages over linear or logarithmic scaling for display of flow cytometry data, a scaling we refer to as "Logicle" scaling. Logicle functions represent a particular generalization of the hyperbolic sine function with one more adjustable parameter than linear or logarithmic functions. Finally, we developed methods for objectively and automatically selecting an appropriate value for this parameter. CONCLUSIONS: The Logicle display method provides more complete, appropriate, and readily interpretable representations of data that includes populations with low-to-zero means, including distributions resulting from fluorescence compensation procedures, than can be produced using either logarithmic or linear displays. The method includes a specific algorithm for evaluating actual data distributions and deriving parameters of the Logicle scaling function appropriate for optimal display of that data. It is critical to note that Logicle visualization does not change the data values or the descriptive statistics computed from them.
LAH
#518
Fukuhara, T., T. Hosoya, S. Shimizu, K. Sumi, T. Oshiro, Y. Yoshinaka, M. Suzuki, N. Yamamoto, L. A. Herzenberg, L. A. Herzenberg and M. Hagiwara (2006). "Utilization of host SR protein kinases and RNA-splicing machinery during viral replication." Proc Natl Acad Sci U S A.
Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.
LAH #519
Wang, D., Carroll, G. T., Turro, N. J., Koberstein, J. T., Kovac, P., Saksena, R., Adamo, R., Herzenberg, L. A., Herzenberg, L. A. and Steinman, L. (2007). "Photogenerated Glycan Arrays Identify Immunogenic Sugar Moieties of Bacillus Anthracis Exosporium." Proteomics 7(2): 180-4.
Using photogenerated glycan arrays, we characterized a large panel of synthetic carbohydrates for their antigenic reactivities with pathogen-specific antibodies. We discovered that rabbit IgG antibodies elicited by Bacillus anthracis spores specifically recognize a tetrasaccharide chain that decorates the outermost surfaces of the B. anthracis exosporium. Since this sugar moiety is highly specific for the spores of B. anthracis, it appears to be a key biomarker for detection of B. anthracis spores and development of novel vaccines that target anthrax spores.
LAH #520
Tung, J. W. and Herzenberg, L. A. (2007). "Unraveling B-1 Progenitors." Curr Opin Immunol.
B-1 cells comprise a small percentage of the B lymphocytes that reside in multiple tissues in the mouse, including the peritoneal and pleural cavities. Functionally, B-1 cells participate in innate immunity by producing the majority of the natural IgM in serum, which protects against invading pathogens before the onset of the adaptive immune response. B-1 cells arise from fetal and neonatal progenitors and are distinct from the adult bone marrow progenitors that give rise to follicular and marginal zone B-2 cells. Recent studies have attempted to delineate the progenitors of B-1 cells from those of B-2 cells. Notably, the identification of CD45R(-/lo)CD19(+) B-1 progenitors and expression of two surface determinants, CD138 and major histocompatibility class II antigens, distinguish developing B-1 cells from B-2 cells.
LAH #521
Yang, Y., Tung, J. W., Ghosn, E. E., Herzenberg, L. A. and Herzenberg, L. A. (2007). "Division and Differentiation of Natural Antibody-Producing Cells in Mouse Spleen." Proc Natl Acad Sci U S A 104(11): 4542-6.
B-1a cells reside in both the peritoneal cavity and the spleen. LPS stimulates splenic B-1a to differentiate to plasma cells producing natural IgM specific for microbial and self antigens. However, there are conflicting views as to whether the B-1a cells divide before this differentiation occurs, and hence how the resident B-1a population is maintained in the spleen. Studies here resolve this dispute in favor of both sides: we show that (some or all) B-1a cells resident in the spleen respond to LPS by differentiating to plasma cells immediately, without dividing; however, we also show that additional B-1a cells immigrate into the spleen after LPS stimulation and divide at least once before differentiating. Importantly, the studies we presently describe reveal the complex cell migration and differentiation events that collectively underlie the rapid production of natural antibodies in response to in vivo LPS stimulation. Thus, the studies present a different view of the roles that B-1a cells play in the early phases of the innate immune response.
LAH #522
Atkuri, K. R., Herzenberg, L. A., Niemi, A. K., Cowan, T. and Herzenberg, L. A. (2007). "Importance of Culturing Primary Lymphocytes at Physiological Oxygen Levels." Proc Natl Acad Sci U S A 104(11): 4547-52.
Although studies with primary lymphocytes are almost always conducted in CO(2) incubators maintained at atmospheric oxygen levels (atmosO(2); 20%), the physiological oxygen levels (physO(2); 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO(2) significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO(2) maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO(2) than at physO(2). This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO(2) than at atmosO(2); and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO(2) that at atmosO(2). Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO(2) likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO(2) because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.
LAH #523
Atkuri, K. R., Mantovani, J. J., Herzenberg, L. A. and Herzenberg, L. A. (2007). "N-Acetylcysteine-a Safe Antidote for Cysteine/Glutathione Deficiency." Curr Opin Pharmacol.
Glutathione (GSH) deficiency is associated with numerous pathological conditions. Administration of N-acetylcysteine (NAC), a cysteine prodrug, replenishes intracellular GSH levels. NAC, best known for its ability to counter acetaminophen toxicity, is a safe, well-tolerated antidote for cysteine/GSH deficiency. NAC has been used successfully to treat GSH deficiency in a wide range of infections, genetic defects and metabolic disorders, including HIV infection and COPD. Over two-thirds of 46 placebo-controlled clinical trials with orally administered NAC have indicated beneficial effects of NAC measured either as trial endpoints or as general measures of improvement in quality of life and well-being of the patients.
LAH #524
Baumgarth, N., Choi, Y. S., Rothaeusler, K., Yang, Y. and Herzenberg, L. A. (2007). B Cell Lineage Contributions to Antiviral Host Responses. Specialization and Complementation of Humoral Immune Responses to Infection. T. Manser. Berlin Heidelberg, Springer-Verlag. 319: 41-61.
B cell responses are a major immune protective mechanism induced against a large variety of pathogens. Technical advances over the last decade, particularly in the isolation and characterization of B cell subsets by multicolor flow cytometry, have demonstrated the multifaceted nature of pathogen-induced B cell responses. In addition to participation by the major follicular B cell population, three B cell subsets are now recognized as key contributors to pathogen-induced host defenses: “marginal zone (MZ)” B cells, B-1a and B-1b cells. Each of these subsets seems to require unique activation signals and to respond with distinct response patterns. Here we provide a brief review on the main developmental and functional features of these B cell subsets. Furthermore, we outline our current understanding of how each subset contributes to the humoral response to influenza virus infection and what regulates their differential responses. Understanding of the multilayered nature of the humoral responses to infectious agents and the complex innate immune signals that shape pathogen-specific humoral responses are likely at the heart of enhancing our ability to induce appropriate and long-lasting humoral responses for prophylaxis and therapy.
LAH #526
Tung, J. W., Heydari, K., Tirouvanziam, R., Sahaf, B., Parks, D. R., Herzenberg, L. A. and Herzenberg, L. A. (2007). "Modern Flow Cytometry: A Practical Approach." Clinics in Laboratory Medicine 27(3): 453–468.
The use of fluorescence-activated cell sorting (FACS) instruments and methods for clinical purposes dates almost to the time that this unique technology was introduced [1,2]. The widespread application of FACS in clinical research and practice really began, however, with the development of monoclonal antibodies that recognized surface proteins or other markers that distinguished functional subsets of peripheral blood lymphocytes from one another. Once this was accomplished, the problem was not whether or not FACS would be used but how to produce the reagents and refine the technology so that clinically significant subsets could be identified, counted, sorted, and even transferred to appropriate recipients. The demonstration that CD4 T-cell counts can be used to monitor HIV disease progression opened the way to the first clinical FACS application [3,4]; the demonstration that stem cells can be sorted and transferred to appropriately pretreated recipients now opens the way to new and constructive FACS uses in the future [5].
LAH #527
Herzenberg, L. A. (2007). "Epitope-Specific Regulation: The Elephant in the Bathtub." Nat Immunol 8(8): 783-6.
Supplemental Material
Tirouvanziam, R., Y. Gernez, et al. (2008). "Profound functional and signaling changes in viable inflammatory neutrophils homing to cystic fibrosis airways." Proc Natl Acad Sci U S A 105(11): 4335-9.
Blood neutrophils recruited to cystic fibrosis (CF) airways are believed to be rapidly killed by resident bacteria and to passively release elastase and other toxic by-products that promote disease progression. By single-cell analysis, we demonstrate that profound functional and signaling changes readily occur within viable neutrophils recruited to CF airways, compared with their blood counterparts. Airway neutrophils have undergone conventional activation, as shown by decreased intracellular glutathione, increased lipid raft assembly, surface mobilization of CD11b+ and CD66b+ granules, and increased levels of the cytoskeleton-associated phospho-Syk kinase. Unexpectedly, they also mobilize to the surface CD63+ elastase-rich granules, usually confined intracellularly, and lose surface expression of CD16 and CD14, both key receptors in phagocytosis. Furthermore, they express CD80, major histocompatibility complex type II, and the prostaglandin D2 receptor CD294, all normally associated with other lineages, which reflects functional reprogramming. This notion is reinforced by their decreased total phosphotyrosine levels, mirroring a postactivated stage, and increased levels of the phospho-S6 ribosomal protein, a key anabolic switch. Thus, we identified a subset of neutrophils within CF airways with a viable but dysfunctional phenotype. This subset provides a possible therapeutic target and indicates a need to revisit current paradigms of CF airway disease.
LAH #530
Sahaf, B., K. Atkuri, K. Heydari, M. Malipatlolla, J. Rappaport, E. Regulier, L. A. Herzenberg and L. A. Herzenberg (2008). "Culturing of human peripheral blood cells reveals unsuspected lymphocyte responses relevant to HIV disease." Proc Natl Acad Sci U S A 105(13): 5111-6.
Recombinant HIV-Tat (Tat) induces extensive apoptosis in peripheral blood mononuclear cells (PBMCs) cultured in typical CO(2) incubators, which are equilibrated with air (21% O(2)). However, as we show here, Tat apoptosis induction fails in PBMCs cultured at physiological oxygen levels (5% O(2)). Under these conditions, Tat induces PBMCs to divide, efficiently primes them for HIV infection, and supports virus production by the infected cells. Furthermore, Tat takes only 2 h to prime PBMCs under these conditions. In contrast, PHA/IL-2, which is widely used to prime cells for HIV infection, takes 2-3 days. These findings strongly recommend culturing primary cells at physiological oxygen levels. In addition, they suggest HIV-Tat as a key regulator of HIV disease progression.