Description and Characterization of a Novel Human Mast Cell Line for Scientiﬁc Study

International Journal of Molecular Sciences

Communication Description and Characterization of a Novel Human Mast Cell Line for Scientiﬁc Study

Arnold S. Kirshenbaum 1,* , Yuzhi Yin 1, J. Bruce Sundstrom 2, Geethani Bandara 1 and Dean D. Metcalfe 1

1 Mast Cell Biology Section, Laboratory of Allergic Diseases, Bethesda, MD 20892, USA; [email protected] (Y.Y.); [email protected] (G.B.); [email protected] (D.D.M.) 2 AIDS Research Review Branch/SRP, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; [email protected] * Correspondence: [email protected]; Tel.: +301-761-6654

Received: 21 August 2019; Accepted: 29 October 2019; Published: 6 November 2019

Abstract: Background: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. Methods: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (β-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. Results: LADR cells were larger and more granulated as seen with Wright–Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. Conclusions: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.

Keywords: mast cell; LAD cells; HIV; allergy; FcεR1; stem cell factor; c-KIT; SOCS; MYC

1. Introduction Mast cells are known to play a major role in innate and acquired immunity. Since 1988, a number of cell lines have been described, including HMC-1 (Human Mast Cell leukemia-1), LAD2 (Laboratory of Allergic Diseases 2), LUVA (Laboratory of University of VirginiA), and ROSA cell lines [1–4]. HMC-1 and LAD2 cell lines were derived from patients with mast cell leukemia. HMC 1.1 and 1.2

Int. J. Mol. Sci. 2019, 20, 5520; doi:10.3390/ijms20225520 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5520 2 of 9 express KIT mutations, while LAD2 cells do not express mutations in KIT. By contrast, LUVA and ROSAKIT WT cell lines were derived from CD34+ cells of non-mastocytosis donors and do not express KIT mutations. ROSAKIT D816V cells do exhibit activating KIT mutations. All cell lines are available for study of preclinical human mast cell biology in lieu of primary mast cell cultures derived from bone marrow, or peripheral or cord blood precursors [5]. The LAD2 mast cell line closely resembles primary humanInt. J. Mol. mast Sci. 2019 cell, 20, x cultures FOR PEER REVIEW which are slow growing, have growth dependence on2 theof 9 presence of stem cell factor (SCF), bear functional surface FcεRI receptors, and have the ability to degranulate to KIT WT immunologicLUVA stimuli. and ROSA LAD2 cell human lines were mast derived cells from have CD34+ been cells maintained of non-mastocytosis in our laboratory donors and do for use and not express KIT mutations. ROSAKIT D816V cells do exhibit activating KIT mutations. All cell lines are distributionavailable worldwide. for study of preclinical human mast cell biology in lieu of primary mast cell cultures derived LAD2from cells bone were marrow, derived or peripheral from CD34or cord+ bloodcells precursors following [5]. marrow The LAD2 aspiration mast cell line of closely a patient with aggressiveresembles mastocytosis primary where human mutationsmast cell cultures in KIT whichwere are notslow identified. growing, have A growth second dependence aspiration on preserved the presence of stem cell factor (SCF), bear functional surface FcεRI receptors, and have the ability to under liquid N2 gave rise to a second mast cell line LAD1, now re-established and characterized and degranulate to immunologic stimuli. LAD2 human mast cells have been maintained in our laboratory which wefor term use and LADR. distribution As will worldwide. be shown, LADR cells share some similarities to LAD2 cells while differing in someLAD2 important cells were aspectsderived from of degranulation, CD34+ cells following surface marrow receptor aspiration expression, of a patient protease with content, gene expression,aggressive and mastocytosis susceptibility where tomutations infection. in KIT were not identified. A second aspiration preserved under liquid N2 gave rise to a second mast cell line LAD1, now re-established and characterized and 2. Resultswhich we term LADR. As will be shown, LADR cells share some similarities to LAD2 cells while differing in some important aspects of degranulation, surface receptor expression, protease content, We firstgene expanded expression, andand susceptibility then characterized to infection. LADR cells after removing them from liquid nitrogen and testing for cell viability. In culture, LADR cells were larger, more granulated, and slower to 2. Results proliferate (Figure1A,B), suggesting a more advanced and mature cell line. LADR granular content of We first expanded and then characterized LADR cells after removing them from liquid nitrogen tryptase was a log-fold higher compared to LAD2 cells (Figure1C). LADR cells stained for granular and testing for cell viability. In culture, LADR cells were larger, more granulated, and slower to chymase asproliferate has been (Figure reported 1A, B), for suggesting LAD2 cellsa more (Figure advanced1D). and Degranulation mature cell line. LADR and βgranular-hex release content surpassed that of LAD2of tryptase cells (Figurewas a log-fold1E). Flowhigher cytometrycompared to LAD2 studies cells confirmed (Figure 1C). theLADR larger cells stained size (FSC) for granular and increased granularitychymase (SSC) as (Figure has been2 A)reported of LADR for LAD2 cells. cells All(Figure LADR 1D). Degranulation cells stained and positive β-hex release forCD117 surpassed and Fc εRI, that of LAD2 cells (Figure 1E). Flow cytometry studies confirmed the larger size (FSC) and increased with increased expression of CD117 and FcεRI when compared to LAD2 cells (Figure2B,C). As shown granularity (SSC) (Figure 2A) of LADR cells. All LADR cells stained positive for CD117 and FcεRI, in Table1,with cell increased surface expression markers showedof CD117 and the Fc addedεRI when presence compared on to LADR LAD2 cells cells (Figure of CD13, 2B,C). CD123,As and complementshown receptors in Table CD1841, cell surface and markers CD195 showed are consistent the added with presence HIV on studies, LADR cells as of will CD13, be shown.CD123, and complement receptors CD184 and CD195 are consistent with HIV studies, as will be shown.

Figure 1. FigureCell 1. proliferation,Cell proliferation, tryptasetryptase expression, expression, chymase chymase expression, expression, degranulation, degranulation, and beta- and beta-hexosaminidasehexosaminidase (β (β-hex)-hex) release release of LADR of LADR (a second (a secondmast cell mastline) and cell laboratory line) and of allergic laboratory diseases of allergic 2 (LAD2) cells. (A) LADR cell numbers (in red) doubled in 3–4 weeks compared with 1–2 weeks for diseases 2LAD2 (LAD2) cells cells.(in black), (A LADR) LADR cells cell appeared numbers to expand (in in red) culture doubled as a more in advanced 3–4 weeks human compared mast cell with 1–2 weeks for LAD2line; (B) cellsWright–Giemsa (in black), staining LADR of cellsLADR appeared cells (×630); to (C expand) LADR cells in culture (in blue)as have amore log-fold advanced higher human mast cell line;granular (B) Wright–Giemsaexpression of tryptase; staining (D) LADR of LADR cells express cells (chymase630); ((inC) red, LADR and cells(E) LADR (in blue) cell β-hex have log-fold × higher granular expression of tryptase; (D) LADR cells express chymase (in red, and (E) LADR cell β-hex release (left panel) was twice the release of LAD2 cells (right panel) following Ag crosslinking alone and with SCF (stem cell factor) enhancement. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 9

Int. J. Mol.release Sci. 2019 (left, 20 panel), 5520 was twice the release of LAD2 cells (right panel) following Ag crosslinking alone 3 of 9 and with SCF (stem cell factor) enhancement.

FigureFigure 2. 2.Flow Flow cytometry cytometry studies studies comparingcomparing LADR with with LAD2 LAD2 cells. cells. (A (A) LADR) LADR cells cells (upper (upper panel) panel) areare larger larger (based (based on on FSC, FSC, horizontal horizontal axis)axis) andand moremore granulated (based (based on on SSC, SSC, vertical vertical axis) axis) when when comparedcompared with with LAD2 LAD2 cells cells (lower (lower panel).panel). (B) LADR cells cells (upper (upper panel) panel) have have higher higher expression expression of of FcεFcRIεRI (horizontal (horizontal axis) axis) and and CD117 CD117 (vertical(vertical axis) when compared compared with with LAD2 LAD2 cells cells (lower (lower panel), panel), and and (C)(C histograms) histograms of of CD117 CD117 (upper (upper panel) panel) andand FcεRI (lower panel) panel) expression expression comparing comparing LADR LADR (in (inred) red) andand LAD2 LAD2 cells cells (in (in black) black) and and consistent consistent withwith resultsresults in B.

TableTable 1. 1.Surface Surface expression expression of of CD CDmarkers. markers. LADR ce cellslls expressed expressed CD13, CD13, CD33, CD33, CD34, CD34, CD63, CD63, CD117, CD117, CD123,CD123, CD133, CD133, CD184, CD184, CD193, CD193, andand CD195,CD195, while LAD2 cells cells expressed expressed CD33, CD33, CD34, CD34, CD63, CD63, CD117, CD117, CD133,CD133, and and CD193 CD193 but but not not CD13, CD13, CD CD 25,25, CD123,CD123, CD184, or CD195. CD195.

CD MarkerCD Marker LADRLADR Cells Cells LAD2 LAD2 Cells Cells CD13 + + CD13 + + −− CD25 CD25 − − −− CD33 + + CD33CD34 + + + + CD34CD63 + + + + + + CD117 + + + + + + CD63CD123 + + + + + − CD117CD133 + + + + + + + CD184 + + CD123 + −− CD193 + + CD133CD195 + + + − CD184Fc εRI + + + + +− + Single multiplicationCD193 sign (+) means 1 log increase+ over negative control, + double sign (+ +) means 2 log increase over negative control, triple sign (+ + +) means 3 log increase over negative control. Negative expression is designated by ( ). CD195 + − − FcεRI + + + + StudiesSingle multiplication with LAD2 cellssign (+) previously means 1 log revealed increase over that negative binding control, of monomeric double sign human (+ +) means IgE to2 FcεRI could alogffect increase HIV infectivity over negative following control, triple upregulation sign (+ + +) of means T- and 3 M-tropiclog increase complement over negative co-receptors control. [6]. Thus, toNegative check forexpression LADR /isLAD2 designated functional by (−). differences, LADR and LAD2 cells were initially incubated with monomericStudies with human LAD2 IgE cells and previously complement revealed receptors that werebinding studied. of monomeric As predicted, human earlier IgE andto Fc higherεRI surfacecould expressionaffect HIV infectivity of CD184 following was noted upregulation for LADR of cells T- and at 48 M-tropic h (Figure complement3A), consistent co-receptors with higher [6]. mRNAThus, expressionto check for LADR/LAD2 at 48 h (Figure functional3B, compared differences, with LADR 3A). When and LAD2 infected cells withwere initially HIV with incubated di fferent tropisms, HIV-BAL, HIV-LAV, and HIV-TYBE infectivity, as measured by p24 assay, were detected in

LADR cells earlier (day 8) than LAD2 cells (day 12), consistent with their diﬀerential surface expression of CD184 (Figure3C). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 9

with monomeric human IgE and complement receptors were studied. As predicted, earlier and higher surface expression of CD184 was noted for LADR cells at 48 h (Figure 3A), consistent with higher mRNA expression at 48 h (Figure 3B, compared with 3A). When infected with HIV with different tropisms, HIV-BAL, HIV-LAV, and HIV-TYBE infectivity, as measured by p24 assay, were Int.detected J. Mol. Sci. in2019 LADR, 20, 5520 cells earlier (day 8) than LAD2 cells (day 12), consistent with their differential4 of 9 surface expression of CD184 (Figure 3C).

FigureFigure 3. 3.HIV HIV infectivity infectivity studiesstudies comparing comparing LADR LADR and and LAD2 LAD2 cells. cells. Followin Followingg monomeric monomeric human human IgE IgEtreatment, treatment, (A (A) )LADR LADR cells cells (upper (upper panels) panels) have have higher higher surface surface expression expression of ofCD184 CD184 (green (green curve curve comparedcompared with with red red curve curve isotype isotype control) control)than than LAD2LAD2 cells (lower panels), panels), consistent consistent with with (B ()B higher) higher CD184CD184 mRNA mRNA expression expression at at 24 24 and and 48 48 h,h, andand ((CC)) detectable infection infection (HIV-p24 (HIV-p24 assay) assay) with with HIV-BAL HIV-BAL (left(left graph), graph), HIV-LAV HIV-LAV (middle (middle graph), graph), and and HIV-TYBEHIV-TYBE (right graph) at at day day 8 8for for LADR LADR cells cells (green) (green) comparedcompared with with day day 12 12 for for LAD2 LAD2 cells cells (purple). (purple). The st standardandard deviations deviations in in 3C 3C for for HIV-BAL, HIV-BAL, LAV, LAV, and and TYBETYBE p24 p24 values values all all range range from from 0.05–0.1% 0.05–0.1% and and areare notnot displayed.displayed.

DiffDifferentialerential gene gene expression expression with with a a heat heat mapmap displaydisplay (Figure 44A)A) showed distinctly distinctly upregulated upregulated genesgenes when when comparing comparing LADR LADR with with LAD2 LAD2 cells. cells. AsAs many as 400 400 genes genes were were shown shown to to be be upregulated upregulated (Figure(Figure4B). 4B). When When compared compared with with LAD2 LAD2 cells, cells, major major canonicalcanonical pathway diffe di ffrences,erences, reflecting reflecting gene gene upregulation,upregulation, were were noted noted for for LADR LADR cells cells (Figure (Figure5 5AA).).Examples Examples of upregulated genes genes included included SOCS SOCS (suppressor(suppressor of cytokineof cytokine signaling) signaling) with its with role inits JAK2 role/STAT3 in JAK2/STAT3 signaling and cellularsignaling myelo andc ytomatosiscellular oncogenemyelocytomatosis (c-MYC) withoncogene its central (c-MYC) role with in regulationits central role of cellin regulation growth, of survival, cell growth, and survival, differentiation and (Figuredifferentiation5B). (Figure 5B).

Int. J. Mol. Sci. 2019, 20, 5520 5 of 9 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 9

Figure 4. Differential gene expression of LADR cells compared with LAD2 cells. (A) Heat map Figure 4. Differential gene expression of LADR cells compared with LAD2 cells. (A) Heat map showing differentially expressed and upregulated genes (red) when comparing LADR to LAD2 showing differentially expressed and upregulated genes (red) when comparing LADR to LAD2 cells. cells. Downregulated genes are shown in blue, genes without significant change are shown in white. Downregulated genes are shown in blue, genes without significant change are shown in white. (B) (B) LADR cells expressed over 400 upregulated genes (red) when compared with LAD2 cells. Genes LADR cells expressed over 400 upregulated genes (red) when compared with LAD2 cells. Genes without significant change are shown in blue. without significant change are shown in blue.

Int. J. Mol. Sci. 2019, 20, 5520 6 of 9 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 of 9

Figure 5. Canonical pathways and examples of gene upregulation in LADR cells. (A) Top canonical Figurepathways 5. Canonical for LADR pathways cells compared and examples with LAD2 of cells,gene andupregulation (B) Suppressor in LADR of cytokine cells. (A signaling) Top canonical (SOCS) pathwaysgenes are upregulated,for LADR cells which compared may contributewith LAD2 to cells, an LADR and (B cell) Suppressor inherentJAK2 of cytokine/STAT3 signaling downregulation (SOCS) genesadvantage. are upregulated, Upregulation which of themay viral contribute oncogene to anMYC LADRmay cell provide inherent an JAK2/STAT3 independent downregulation reason for cell advantage.proliferation, Upregulation survival, and of ditheﬀerentiation. viral oncogene Upregulated MYC may genes provide are highlighted an independent in red. reason Black for arrows cell proliferation,designate pathways survival, of and interaction differentiation. between Upregulated signal transduction genes are elements,highlighted blue in red. arrows Black designate arrows designatepathways ofpathways particular of interestinteraction in the between use of this signal cell line,transduction T-arrowrefers elements, to inhibition. blue arrows designate pathways of particular interest in the use of this cell line, T-arrow refers to inhibition. 3. Discussion 3. DiscussionIn 2003, our laboratory published a report of the LAD2 human mast cell line which oﬀered a uniqueIn 2003, opportunity our laboratory to examine published the biologya report ofof humanthe LAD2 mast human cells andmast our cell groupline which has made offered this a uniquecell line opportunity available to to researchers examine the everywhere. biology of human This area mast of cells research and our has group gradually has made matured, this cell and line the available to researchers everywhere. This area of research has gradually matured, and the current

Int. J. Mol. Sci. 2019, 20, 5520 7 of 9 current interest in disease phenotypes with gain-of-function genetic lesions led to the question whether characterization of additional mast cell lines may show differences in gene expression and provide insight into upregulation and control of mast cell activation, proliferation, and chemotaxis. We thus expanded and characterized LADR cells and compared this line to existing LAD2 cells in our laboratory. LADR cells were larger and more granulated as evidenced by histochemistry and flow cytometry and were slower to proliferate in culture (Figures1 and2), suggesting a more advanced and mature cell line. LADR cells, similar to their LAD2 counterparts, survived for weeks in the absence of SCF, but did not proliferate. Degranulation and β-hex release surpassed that of LAD2 cells initially in culture. The LADR granular content of tryptase was a log-fold higher (Figure1), chymase was similarly expressed, and flow cytometry studies (Figure2) showed consistent but increased dual staining for CD117 and FcεRI. We previously reported that incubation with monomeric IgE may affect LAD cell expression of CD4 and co-receptors [6]. Functional differences were predicted when earlier and higher expression of CD184 and CD193 was noted for LADR cells following monomeric human IgE incubation (Figure3). Thus, when LADR cells were infected with HIV with di fferent tropisms, HIV-BAL, HIV-LAV, and HIV-TYBE infectivity, as measured by p24 assay, were detected earlier than LAD2 cells, consistent with their differential surface expression of complement receptors. Previous flow cytometry experiments using CD184 antibody may not have been sensitive enough to detect low level expression of CD184 on LADR at 24 h in the presence of IgE, in contrast to detection of CD184 expression using newer conjugates and LSRFortessa flow cytometry. Gene analysis comparing LADR with LAD2 cells showed a large differential expression of upregulated genes (Figure4) and canonical pathway differences (Figure5), which are now being investigated for physiologic importance and impact on mast cell functions. Our purpose in studying additional LAD cell lines was to compare the characteristics of LADR with those published for LAD2 and determine advantages for using one cell line over the other. To this end, LADR cells offer clear advantages depending on both the research at hand and the human mast cell biology questions being addressed. Compared with LAD2 cells, and other human mast cell lines available, we believe that LADR cells appear to resemble primary human cell cultures, with higher β-hex release upon FcεRI crosslinking, and a higher cellular content of tryptase perhaps due to their slower proliferation rates. HMC 1.1 and 1.2 cells lack functional FcεRI and ROSAKIT WT cells lack chymase expression. Chymase is an efficient Ang II (angiotensin II)-forming enzyme and has been implicated in a wide variety of human diseases that also implicate its many other protease actions. LADR and LAD2 cells do not harbor mutations in KIT, expressed by as much as 80% of patients with systemic mastocytosis (SM), representing a disadvantage of this cell line for studying emerging therapeutics directed at KIT D816V. It should be recognized, however, that KIT-independent pathways and pro-oncogenic hits and lesions may be responsible for disease progression in advanced SM [5]. Thus, for example, due to upregulation of the viral oncogene c-MYC and expression of surface antigens CD13, CD33, CD34, CD117, CD123, and other markers aberrantly expressed on malignant mast cells and/or their progenitors, LADR cells may indeed be a tool for studying cell growth and regulation in advanced SM. The ability of human mast cells to act as a latent reservoir for HIV, as shown in vivo and with LADR cells, may be the best evidence that the LADR human mast cell line has wide-ranging utility and advantages well beyond the study of SM.

4. Materials and Methods

4.1. LAD Cell Cultures LAD cells were maintained in StemPro serum free media with nutrient supplement, glutamine, penicillin/streptomycin, and 100 ng/mL rhSCF as described [7]. Hemi-depletions were performed weekly. Cell proliferation was examined by measuring total cell numbers weekly following staining with Kimura stain. Int. J. Mol. Sci. 2019, 20, 5520 8 of 9

4.2. β-Hex Release Studies β-hex release was assayed following crosslinking of FcεRI as described [8]. Brieﬂy, LADR and LAD2 cells were incubated overnight with 100 ng/mL human myeloma IgE (BD PharMingen, San Diego, CA, USA), biotinylated by the NIAID core facility, followed by crosslinking with 125 ng/mL streptavidin (SA) alone or in combination with rhSCF (Sigma-Aldrich, St. Louis, MO, USA) [9]. β-hex was reported as a percentage of total cell contents released.

4.3. Flow Cytometry Studies LADR and 2 cells were harvested, washed, and incubated with Aqua for to assess viability. Following additional washing, cells were incubated with either CD13, CD25, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, CD195, or FcεRI and examined. Alternatively, cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After permeabilization with cold methanol for 1 h on ice, cells were stained with anti-tryptase for 30 min. For intracellular chymase, cells were fixed and permeabilized with Foxp3 staining buffer set (eBioscience, San Diego, CA, USA) and stained with anti-chymase for 30 min. All analyses were performed on at least 10,000 cells. All cells were analyzed by LSRFortessa flow cytometry.

4.4. Gene Arrays LADR and 2 cells were harvested, and total RNA was extracted from 1 106 LAD2 cells using the × RNeasy Plus RNA isolation kit (Qiagen, Germantown, MD, USA) as described [10]. Approximately 1 µg of total RNA was reverse transcribed (RT) using the SuperScript III First-Strand synthesis system (Invitrogen, Grand Island, NY, USA) with random hexamer primers. cDNA and expression arrays comparing LADR with LAD2 gene expression were then prepared using RT-PCR according to manufacturers’ recommendations. Sequencing data was analyzed using Sequencher (Version 4.5, Softgenetics, State College, PA, USA).

4.5. HIV Infectivity LADR and 2 cells were ﬁrst incubated overnight with/without monomeric human IgE, washed, and examined for complement receptor expression over 96 h as previously described [6]. Stimulated cells were also examined for receptor mRNA expression over 96 h. Infectivity in cultured LADR and LAD2 cells with HIV-BAL, HIV-LAV, and HIV-TYBE was measured by assaying p24 expression [11].

5. Conclusions LADR cells when compared to LAD2 cells have a slower proliferation rate, are larger, and more granulated. LADR cells have increased FcεRI/CD117 and tryptase expression and express chymase. LADR cells have diﬀerent gene expression which is currently being investigated. Following monomeric human IgE treatment, LADR express CD184 and earlier infectivity with HIV-BAL, HIV-LAV, and HIV-TYBE. Thus, while LADR and LAD2 cells share many characteristics, in some situations, one cell line may have an advantage over the other.

Author Contributions: Conceptualization, A.S.K. and D.D.M.; Methodology Y.Y., J.B.S., G.B.; Formal Analysis, A.S.K., J.B.S.; Resources, D.D.M., J.B.S.; Writing—Original Draft Preparation, A.S.K.; Writing—Review & Editing A.S.K. and D.D.M. Funding: This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Conﬂicts of Interest: The authors declare no conﬂict of interest. Int. J. Mol. Sci. 2019, 20, 5520 9 of 9

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