CD99 regulates CXCL12-induced chemotaxis of human plasma cells

Immunology Letters 168 (2015) 329–336

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Immunology Letters journal homepage: www.elsevier.com/locate/immlet

CD99 regulates CXCL12-induced chemotaxis of human plasma cells Minchan Gil a,1 , Hyo-Kyung Pak a,d,1 , A-Neum Lee a , Seo-Jung Park a , Yoonkyung Lee a , Jin Roh d , Hyunji Lee a , Yoo-Sam Chung c , Chan-Sik Park a,b,d,∗ a

Cell Dysfunction Research Center, University of Ulsan College of Medicine, Seoul, South Korea Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea Department of Otolaryngology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea d Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea b c

a r t i c l e

i n f o

Article history: Received 22 June 2015 Received in revised form 8 October 2015 Accepted 23 October 2015 Available online 6 November 2015 Keywords: CD99 Plasma cell Migration Extracellular signal-regulated kinase

a b s t r a c t Migration of plasma cells (PCs) is crucial for the control of PC survival and antibody production and is controlled by chemokines, most importantly by CXCL12. This study investigated the role of CD99 in CXCL12-induced PC migration. Among B cell subsets in the tonsils, CD99 expression was highest in PCs. CD99 expression increased during in vitro differentiation of germinal center B cells and was highest in PCs. CD99 engagement reduced chemotactic migration of PCs toward CXCL12 and reduced extracellular signal-regulated kinase (ERK) activation by CXCL12. An ERK inhibitor reduced CXCL12-mediated chemotactic migration, which suggests that ERK has a critical role in migration. CD99 engagement did not influence apoptosis, differentiation, or antibody secretion of PCs. We propose a novel role of CD99 in PCs that suppresses ERK activation and chemotactic migration of these cells. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Plasma cells (PCs) are the final effecter of humoral immunity, which produces highly specific protective antibodies (Abs) against fatal pathogens. Naïve B cells primed with a T-dependent antigen enter the germinal center (GC) to undergo affinity maturation of the antigen receptor and differentiate into PCs. Early PCs generated in the GC enter the circulation and eventually reside in survival niches in bone marrow (BM) or other tissues. Migration of PCs to survival niches is crucial for the maturation of PCs and associated humoral immunity [1,2]. This PC migration is controlled by chemokine–chemokine receptor axes, including CXCL12-CXCR4 [3–5], CXCL9/10/11-CXCR3 [6], CCL25-CCR9, and CCL28-CCR10 [7–9], according to the destination of PCs. Characterization of the mechanism involved in chemotactic migration of

Abbreviations: Ab, antibody; BM, bone marrow; ERK, extracellular signalregulated kinase; GC, germinal center; IgG, immunoglobulin G; IRB, Institutional Review Board; IL, interleukin; MFI, median fluorescence intensity; PBS, phosphatebuffered saline; PC, plasma cell; PRDM1, PR domain zinc finger protein 1; PI, propidium iodide; SD, standard deviation. ∗ Corresponding author at: Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 388-1, Pungnap-dong, Songpa-gu, Seoul 138-736, Republic of Korea. Fax: +82 10 8940 3669. E-mail address: [email protected] (C.-S. Park). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.imlet.2015.10.015 0165-2478/© 2015 Elsevier B.V. All rights reserved.

PCs is essential to elucidate the survival and function of PCs, and to harness humoral immunity. Notably, studies of CXCR4-knockout mice showed that the CXCL12-CXCR4 axis is crucial for PC homing to BM and humoral immunity [3–5]. However, the detailed regulatory mechanism underlying the response of migratory PCs to CXCL12 remains to be defined. CD99 is a 32 kDa type I transmembrane glycoprotein that is broadly expressed in humans and primates. CD99 is strongly expressed in a particular subtype of cells and is involved in essential functions including apoptosis [10–15], adhesion [11,16–19], differentiation [20–23], and protein trafficking [24–27]. Recently, the role of CD99 in leukocyte migration has been increasingly reported. CD99 expression is indispensable for the migration of various hematopoietic cells including monocytes [28], neutrophils [29], and CD34+ cells [30]. CD99 is also involved in the migration and invasiveness of tumor cells [31–33]. Previous studies uncovered some signaling pathways that operate downstream of CD99 [12–14,16,18,19,26,27,31–35]. Specifically, CD99 induces extracellular signal-regulated kinase (ERK) activation in breast cancer cells [33], osteosarcoma cells [22], and Jurkat lymphoblastic leukemia cells [34]. However, the role of the CD99-dependent signaling pathway in chemotactic migration of PCs has not been reported. Here, we investigated the expression of CD99 in B cell subsets of human tonsils and found that CD99 expression was highest in tonsillar PCs. During in vitro differentiation of GC-B cells, CD99 expression increased and was highest in PCs. Ab-induced

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engagement of CD99 retarded chemotactic migration of PCs toward CXCL12, suggesting that CD99 plays an important role in the migration of PCs. 2. Material and methods 2.1. Cells, Abs, and reagents Mouse L cells expressing CD40 ligand were cultured in RPMI1640 medium. HS-5 cells were cultured in DMEM. All media were supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 ␮g/ml streptomycin (Gibco BRL, Eggenstein, Germany). Anti-CD99 monoclonal Abs YG32 and DN16 (immunoglobulin G (IgG)1 ) were purchased from Dynona Inc. (Seoul, Republic of Korea). Anti-CD99 and anti-CD138 Abs used for immunohistochemistry were purchased from Dako (Glostrup, Denmark). Anti-mouse IgG divalent F(ab) fragments were purchased from Jackson ImmunoResearch (Westgrove, PA, USA). Mouse IgG1 (MOPC31C; used as an isotype control) was purchased from Sigma (St Louis, MO, USA). Goat anti-human Ig(H + L)-UNLB and goat anti-human Ig(H + L)-HRP were purchased from Southernbiotech (Birmingham, Alabama, USA). Human reference serum was purchased from Bethyl Laboratories (Montgomery, TX, USA). All other Abs used for flow cytometric analysis were purchased from BD Biosciences (San Jose, CA, USA). Recombinant human CXCL9, CXCL12, and interleukin (IL)-21 were purchased from Peprotech (Rocky Hill, NJ, USA). IL-2 was provided by Professor J. Choe (College of Medicine, Kangwon National University, ROK). Ficoll-PaqueTM was purchased from GE Healthcare (Waukesha, WI, USA). 2.2. Purification of tonsillar mononuclear cells and flow cytometric analysis Human tonsils were obtained from remainder tissues of therapeutic tonsillectomy and handled in accordance with an Institutional Review Board (IRB)-approved protocol (2013–0864). The need for informed consent was waived by the IRB on the following basis: (1) there was no additional risk to the participants; remainder tissue samples from routine tonsillectomy and pathologic examinations were used for the in vitro experiment to study general phenomena; and (2) patient identities were anonymized and completely unlinked with unique identifiers. Tonsillar mononuclear cells were isolated by mechanical disruption, followed by FicollPaqueTM density gradient centrifugation as previously described [36]. For flow cytometric analysis, tonsillar mononuclear cells were stained for CD3, CD38, IgD, and CD99. Cells were divided into CD3+ (T cells) and CD3− (mostly B cells). The CD3-population was further subdivided into B cell subsets according to IgD and CD38 expression as follows [36–39]: IgD+, naïve B cells; IgD − CD38−, memory B cells; IgD − CD38+, GC-B cells; and IgD − CD38++, PCs. CD99 expression in each B cell subset and in T cells was measured with the DN16 Ab and analyzed using FlowJo software (Ashland, OR, USA). Expression of CD99 was compared using the median fluorescence intensity (MFI). Data are presented as the means of triplicate experiments ± standard deviation (SD).

silanized charged slides, allowed to dry for 10 min at room temperature, and incubated at 65 ◦ C for 20 min. After deparaffinization, heat-induced epitope retrieval using standard Cell Conditioning 1 was performed for 24 min. Subsequently, samples were labeled with primary anti-CD99 (1.20E + 08, 1:200, Dako) and anti-CD138 (MI-15, 1:100, Dako) Abs using an automated immunostaining system with the OptiView DAB Detection Kit (Ventana Medical Systems). Immunostained sections were counter-stained with hematoxylin. 2.4. Preparation and differentiation of human tonsillar GC-B cells GC-B cells were purified from tonsillar mononuclear cells by magnetic-activated cell sorting (MiltenyiBiotec, Auburn, CA, USA), as described previously [36,40]. Briefly, tonsillar mononuclear cells were incubated with anti-IgD, anti-CD44, and anti-CD3 Abs for 15 min on ice, washed and then, incubated with goat anti-mouse IgG 1 microbeads for 15 min on ice. After the final wash, the cells were loaded on a magnetic cell separation column and the negative fraction was collected. The purity was greater than 95%, as assessed by expression of CD20 and CD38. GC-B cells were differentiated as previously described [41] with some modifications. GC-B cells (2 × 105 cells/well) were cultured in 24-well plates in the presence of irradiated mouse fibroblast L cells expressing the CD40 ligand [42] (2 × 104 cells/well), IL-2 (30 U/ml), and IL-21 (30 ng/ml) for 4 days. Cultured cells were harvested and counted, and then 1 × 105 cells were cultured in the presence of irradiated HS-5 cells, IL-2 (30 U/ml), and IL-21 (30 ng/ml) for 3 days. For CD99 engagement, an anti-CD99 Ab YG32 (10 ␮g/ml) was added to cultures with anti-mouse IgG divalent F(ab) fragments in order to crosslink Abs. 2.5. Reverse transcription (RT)-PCR To examine mRNA expression, total RNA was extracted from cells at day 0, 4, and 7 of in vitro differentiation using a NucleoSpin kit (Macherey-Nagel, Düren, Germany). One microgram of RNA was transcribed using an IScript DNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Real-time quantitative PCR was performed using a Step-OneTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a Power SYBR-Green PCR kit (Applied Biosystems). Fold induction was calculated with the 2(−C(T)) method [43], using expression of the ribosomal protein S18 as the reference. The primers used are listed in Table 1. For expression analyses of CD99 type I and II, cDNA was amplified using specific primers for CD99 type I and type II, and analyzed by gel electrophoresis as described previously [18,33]. 2.6. Chemotaxis assay Chemotaxis was determined using filters (pore size, 5 ␮m) for the transwell migration assay (Corning, Tewksbury, MA, USA) according to the manufacturer’s instructions. In vitro-generated PCs were treated with an anti-CD99 Ab or control for 24 h. Then, 1 × 105 cells were placed in the upper migration chamber, with 100 nmol/L CXCL12 in the lower chamber. After 5 h of incubation, the number of cells that had migrated into the lower chamber was counted using Accuri C6 flow cytometry (BD Biosciences).

2.3. Immunohistochemistry 2.7. Intracellular staining of phospho-ERK Immunohistochemical staining was performed on selected serial sections of formalin-fixed, paraffin-embedded tissue blocks. Each staining was performed using an auto immunostainer BenchMark XT (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions and using the reagents supplied with the kit. In brief, 4 ␮m-thick sections were mounted on

Intracellular protein staining was performed according to BD Phosflow Protocol III. Briefly, after CXCL12 stimulation, cells were fixed by adding pre-warmed BD Cytofix buffer. Fixed cells were permeabilized by incubation in chilled BD Perm Buffer III for 30 min. After permeabilization, cells were incubated with an Alexa Fluor®

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Table 1 Primers used for real-time PCR. Gene

Forward primer

Reverse primer

PRDM1 BCL6 CD99 S18

5’-ATCTCAGGGCATGAACAAGG-3’ 5’-CTGCAGATGGAGCATGTTGT-3’ 5’-AGAGCAGAGATGGAGGCCTTCT-3’ 5’-TTTGCGAGTACTCAACACCAACA-3’

5’-ATGGGAAGGCTATGCAAACA-3’ 5’-TCTTCACGAGGAGGCTTGAT-3’ 5’-CCCAGCAACAAGCAAAGCA-3’ 5’-CCTCTTGGTGAGGTCAATGTCTG-3’

488 mouse anti-ERK1/2 (pT202/pY204) Ab (BD Biosciences, catalogue number 554.655) for 1 h. 2.8. ELISA Nunc-ImmunoTM MicroWellTM 96-well plates (Thermo Scientific, Marietta, OH, USA) were coated with 10 ␮g/ml goat anti-human Ig(H + L)-UNLB prepared in phosphate-buffered saline (PBS) overnight at 4 ◦ C, washed with PBS containing 0.05% Tween 20 (PBST), and blocked with 1% bovine serum albumin for 1 h at room temperature. Plates were washed with PBST and incubated with culture supernatant and 5-fold serial dilutions of human reference serum (from 250 ng/ml) in 100 ␮l of PBST for 1 h at room temperature. Plates were washed with PBST, incubated with goat anti-human Ig(H + L)-HRP prepared in PBST for 1 h at room temperature, washed with PBST, and developed by adding TMB substrate (PIERCE, Rockford, IL, USA). The reaction was stopped with 2% oxalic acid, and absorbance at 415 nm was measured using a SunriseTM microplate reader (TECAN, Männedorf, Switzerland). 2.9. Apoptosis assay Apoptosis was analyzed using an apoptosis detection kit (BD Biosciences). Cells (1 × 106 ) were suspended in binding buffer and incubated with Annexin V-FITC and propidium iodide (PI) for 15 min at room temperature in the dark. Samples were then measured using a FACSCalibur flow cytometer and analyzed using CellQuest-Pro software (BD Biosciences).

interfollicular area of lymph nodes. CD99 expression was higher in these cells than in GC and naïve B cells in the mantle zone. These CD99 immunopositive cells in the interfollicular area includes PC, which was confirmed by CD138 immunohistochemistry of serial sections (Fig. 1C). 3.2. Expression of CD99 increases during in vitro differentiation of GC-B cells Because CD99 expression was highest in PCs, we investigated changes in CD99 expression during the differentiation of GC-B cells into PCs. We differentiated GC-B cells in vitro using a combination of cytokines. GC-B cells were initially cultured with IL-2 and IL-21 in the presence of mouse fibroblast L cells expressing the CD40 ligand for 4 days and subsequently with IL-2 and IL-21 on the human BM cell line HS-5 for 3 days (Fig. 2A). Flow cytometric analysis of the surface expression of CD38 and CD20 and real-time PCR analysis of PR domain zinc finger protein 1 (PRDM1, also known as BLIMP1) and BCL6 indicated that GC-B cells successfully differentiated into PCs in this culture condition (Fig. 2B and C). CD99 surface expression was increased at day 4 and was highest at day 7 (Fig. 2D). CD99 has two major subtypes, I and II, which are functionally distinct from each other [18,33,46]. RT-PCR analysis with CD99 type I-specific primers showed that CD99 type I was transcriptionally upregulated in the first 4 days of culture (Fig. 2E). However, we did not detect expression of CD99 type II by RT-PCR (data not shown). These data show that CD99 expression increased during GC-B cell differentiation and was highest on PCs, verifying the CD99 expression data obtained in tonsillar mononuclear cells shown in Fig. 1.

2.10. Statistical analysis 3.3. CD99 signaling retards chemotaxis towards CXCL12 All experiments were repeated three times, unless indicated otherwise in the figure legends. Data are presented as the mean ± SD. Differences between groups were analyzed with the Student’s t-test. A P-value <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism, version 3.02 (GraphPad, La Jolla, CA, USA). 3. Results 3.1. CD99 is differentially expressed among human tonsillar B cell subsets Expression of CD99 in hematopoietic cells was analyzed previously in several reports [44,45]. However, specific expression of CD99 in each tonsillar B cell subset and in PCs has not been assessed quantitatively. We measured CD99 expression in tonsillar B cell lineage subsets by multicolor flow cytometry. Among CD3-cells, CD99 expression in subsets of tonsillar B lineage cells was further examined by gating these cells according to CD38 and IgD expression (Fig. 1A): IgD + CD38−, naïve B cells; IgD − CD38−, memory B cells; IgD − CD38+, GC-B cells; and IgD − CD38++, PCs. Among B cell lineage subsets, CD99 expression was highest in PCs and lowest in naïve B cells (Fig. 1B). The CD99 expression pattern in the B cell subsets was consistent among the three donors (Fig. 1B). We also analyzed CD99 expression in tonsillar sections by immunohistochemistry. CD99 was highly expressed in cells in the

The strong expression of CD99 in PCs suggests that CD99 has an important function in these cells. Recently, the involvement of CD99 in the migration of blood cells has been increasingly reported [28–30]. Human early PCs are migratory and responsive to the chemokine CXCL12 [47,48]. PCs obtained by in vitro differentiation are also reportedly responsive to CXCL12 [23]. We tested whether PCs generated in our in vitro system had a chemotactic response to the PC-attracting chemokines CXCL12 and CXCL9. In a transwell migration assay, PCs exhibited a chemotactic response to CXCL12, but a negligible response to CXCL9 (Fig. 3A). Next, we examined the expression of the CXCL12 receptor, CXCR4, on PCs. CXCR4 expression was higher on PCs at day 7 of culture than on GC-B cells at day 0 (Fig. 3B). These results indicate that PCs in our in vitro development system had a similar CXCR4 expression pattern as human plasmablasts, as previously reported [47,48], and can thus respond to CXCL12. We also examine the role of ERK activity in CXCL12-induced transwell migration using an ERK inhibitor. The ERK inhibitor U0126 significantly reduced transwell migration of PCs (Fig. 3C) but did not affect the cell viability (data is not shown). These results show that CXCL12-induced chemotaxis is dependent on ERK activity in PCs. CD99 controls ERK signaling [22,33,34]. To address the function of CD99 in the chemotactic migration of PCs, PCs were pretreated with an anti-CD99 Ab for engagement of CD99 for 20 h and subjected to the transwell migration assay with CXCL12. CD99 engagement significantly reduced

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Fig. 1. Analysis of CD99 expression in tonsillar B cell subsets. (A) Tonsillar mononuclear cells were stained with anti-CD3, anti-CD38, and anti-IgD Abs, and analyzed by flow cytometry. Non-T cell subsets (CD3−) were distinguished according to expression of IgD and CD38: IgD + CD38−, naïve B cells (NB); IgD − CD38−, memory B cells (MB); IgD − CD38+, GC-B cells; IgD − CD38++, PCs. (B) CD99 surface expression on each B cell subset was analyzed by flow cytometry. The CD99 expression level on each subset from three donors is presented as the normalized MFI (MFI of NB set to 1) in the graph. (C) CD99 expression in tonsillar sections. Left panel, CD99 is highly expressed in cells in the interfollicular area of lymph nodes. Expression of CD99 is higher in these cells than in GC and naïve B cells in the mantle zone (MZ). Right panel, PCs were stained for CD138 on serial sections. Left panel: CD99, ×100. Right panel: CD138, ×100.

transwell migration by 39% (Fig. 3D). Next, we investigated ERK phosphorylation by CD99 engagement. Intracellular flow cytometric analysis showed that CD99 suppressed CXCL12-mediated ERK phosphorylation in PCs (Fig. 3E). These data suggest that CD99 signaling reduces CXCL12-mediated chemotactic migration of PCs by suppressing ERK activity.

PRDM1 expression level (Fig. 4D) upon CD99 engagement. Because CD99 is involved in protein transport [24–27], we also examined the role of CD99 in the secretion of immunoglobulins by PCs. The amount of IgG secreted into the culture media were not significantly changed by CD99 engagement (Fig. 4E). 4. Discussion

3.4. CD99 engagement does not affect apoptosis, differentiation, or Ab production of PCs We examined apoptosis, differentiation, and secretion, which are reportedly consequences of CD99 signaling in other cell types. Engagement of CD99 induces apoptosis in various cells [10–15]. Therefore, we were curious about whether CD99 engagement induces apoptosis in PCs. Apoptosis was assessed by annexin V/PI staining, and annexin V+ cells were considered to be apoptotic (Fig. 4A). The ratio of apoptotic cells was not significantly changed at 3, 6, or 12 h after CD99 engagement. In Jurkat cells, anti-CD99 antibody increase Fas expression [13]. However, CD99 engagement did not change FAS expression level in PC (Fig. 4B). These data show that CD99 engagement does not induce apoptosis of PCs. CD99 leads to PC differentiation in a Hodgkin/Reed-Sternberg cell line by upregulating PRDM1 [23]. We examined the effect of CD99 engagement on PC differentiation by flow cytometric measurement of CD38 and CD20 surface expression and the PRDM1 expression level. There was no significant difference in the number of PCs (Fig. 4C) or the

The regulatory mechanism of the CXCR4-CXCL12 axis in PCs is crucial for humoral immunity. CXCL12-mediated chemotactic migration is essential for the homing of PCs to various destinations, including BM. However, study of the chemotactic migration of PCs is limited, partly because of its rarity in peripheral blood and BM. In this study, we examined the regulatory role of CD99 in the chemotactic migration of PCs, using PCs acquired by in vitro differentiation. We investigated the expression and function of CD99 on human tonsillar B cell lineage cells and PCs. CD99 can mediate diverse cellular process such as adhesion, transendothelial migration, differentiation, and apoptosis of leukocytes. CD99 expression has been studied in B cell precursors during human B cell development stages [44,49]. However, the expression and function of CD99 in the late stage of B cell differentiation into PCs has not been studied comprehensively. We showed quantitatively that CD99 expression is higher in PCs than in all other B cell subsets in human tonsils for the first

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Fig. 2. Expression of CD99 during in vitro GC-B differentiation. (A) GC-B cells were differentiated into PCs in vitro. Isolated GC-B cells from tonsils were cultured with IL-2 and IL-21 in the presence of mouse L cells expressing CD40 ligand for 4 days, and subsequently with IL-2 and IL-21 on HS-5 cells for 3 days. Differentiation was determined by the surface expression of CD20 and CD38 using flow cytometry. (B) Expression levels of the transcription factor PRDM1 and BCL6 were measured by real-time PCR in cells at days 0, 4, and 7 of in vitro culture. (C) Surface expression of CD99 was measured by flow cytometry at days 0, 4, and 7 of culture. (D) Expression of CD99 type I mRNA was measured by RT-PCR at days 0, 4, and 7 of differentiation culture. The graph shows relative CD99 expression measured in three independent experiments.

Fig. 3. The chemotactic responsiveness of PCs is reduced by CD99 engagement. (A) The chemotactic responsiveness of PCs to CXCL9 and CXCL12 was measured by a transwell migration assay. PCs (1 × 105 ) were placed in the upper chamber, with 100 nM CXCL9 or CXCL12 in the lower chamber. After incubation for 5 h, the number of cells in the lower chamber was counted by Accuri C6 flow cytometry. Relative migration was determined by the number of the migrated cells normalized to untreated cell. (B) Flow cytometric analysis shows that CXCR4 expression is higher in PCs (culture day 7) than in GC-B cells (culture day 0). (C) Inhibition of ERK reduces CXCL12-induced transwell migration. PCs were pretreated with the ERK inhibitor U0126 and applied to the upper chamber for the transwell migration experiment. (D) CD99 engagement reduces CXCL12-induced transwell migration. PCs (1 × 105 ) treated with an anti-CD99 Ab YG32 or control Ab were placed in the upper chamber for the transwell migration assay. Relative migration was determined by the number of the migrated cells normalized to control (E) Intracellular phosphor-ERK staining shows that CD99 engagement suppresses CXCL12-induced ERK phosphorylation. PCs treated with an anti-CD99 Ab for 20 h were stimulated with CXCL12 for 2 min. Cells were fixed, stained with an phosphor-ERK specific Ab, and measured by flow cytometry. The expression level of phospho-ERK in CD99-engaged PC and control is presented in representative diagram and graph from three independent experiments.

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Fig. 4. CD99 engagement does not affect apoptosis, differentiation, or Ab production. (A) Apoptosis was measured by PI/Annexin V staining at 3, 6, and 24 h after CD99 engagement. Cells (0.5 × 106 ) were plated and incubated with an anti-CD99 Ab or control. The numbers of Annexin V+ cells (apoptotic population) and PI-/Annexin V-cells are presented in the graphs. (B) Surface expression of FAS was measured by flow cytometry in CD99-engaged and control (C) The number of PCs (CD38+/CD20−) was measured after CD99 engagement or control treatment for 3 days (from day 4 to 7 of culture) and 1 day (from day 6 to 7 of culture). A representative flow cytometric profile is presented and the absolute numbers of PCs are shown in the graph. (D) Expression of PRDM1 was measured after CD99 engagement or control treatment for 3 days, from day 4 to 7 of culture. (E) The amount of human IgG secreted into the culture supernatant was measured by ELISA at day 7 of culture, after CD99 engagement from day 4 of culture. The amount of IgG secreted into the culture supernatant is presented as the concentration of IgG, and the relative amount of IgG divided by the cell number is presented.

time (Fig. 1). PCs are professional Ab-secreting cells and express a relatively small number of essential genes to support their function, such as PRDM1, IRF4, CD38, and CD138, and do not express most molecules characteristic of B cells, including the lymphocyte common antigen CD45 [50,51]. Strong CD99 expression in PCs indicates that CD99 has a functional role in these cells. CD99 is involved in apoptosis, adhesion, differentiation, secretion, and migration in various cancer cells and leukocytes. CD99 is reportedly involved in PC differentiation of Hodgkin/Reed-Sternberg cells by modulating PRDM1 expression [21]. However, we could not detect any difference in PRDM1 expression, expression of the surface marker CD38, or Ab secretion in PCs upon CD99 engagement. In addition, CD99 engagement induces apoptosis in thyomocytes [10], leukemic cells

[13,14], Ewing sarcoma cells [11,15], and TEL/AML1+ acute lymphoblastic leukemia and normal B cell precursors [12]. The lack of an evident effect of CD99 engagement on PC apoptosis suggests that CD99 has cell-specific functions. CD99 engagement also did not influence IgG secretion. In vitro-differentiated human PCs are reportedly migratory when stimulated with CXCL12 [23]. However, the detailed regulatory mechanism underlying CXCL12-mediated migration of human PCs has not been reported. We used ERK inhibitor treatment to determine that ERK activity is involved in CXCL12-mediated transwell migration of PCs. CXCL12 activates ERK in most cells [52], but the requirement of ERK activity for chemotactic migration is cell type-dependent. Inhibition of ERK activation is not involved

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in CXCL12-mediated transwell migration of B cell lines [53]. The involvement of ERK in CXCL12-mediated migration of PCs is a novel finding. In our experiment, CD99 engagement by an agonistic Ab for 20 h retarded CXCL12-mediated transwell migration and reduced ERK phosphorylation. Our results suggest that CD99-engaged PCs become less sensitive to CXCL12 due to inhibition of ERK phosphorylation. The detailed signaling pathway that reduces CXCL12-induced ERK phosphorylation remains to be determined in a future study. CD99 has two isoforms produced by alternative splicing. CD99 type I is the full-length and most abundant isoform [18]. CD99 type II has the truncate c-terminal cytoplasmic domain generated by alternative splicing. The CD99 type I and II have distinct function and differential expression from each other in various types of cells [18,33,46]. In B cells, CD99 Type II inhibits homotypic adhesion of B cells, whereas activation of type I promotes the adhesion process [18]. In PC, we only detected the CD99 type I transcript. This result suggest that CD99 function in PC mainly comes from CD99 type I. This study suggests a novel role for CD99, namely, regulation of CXCL12-dependent chemotactic migration of PCs. The CXCL12CXCR4 axis regulates the trafficking of many types of cells including hematopoietic stem cells, neutrophils, and PCs. Many CXCR4 inhibitors are in clinical trials or approved therapeutic agents for cancers, autoimmune diseases, and stem cell mobilization [54]. CXCL12-dependent chemotactic migration of PCs determines their localization and function. CXCR4-knockout mouse studies revealed elevated numbers of PCs in blood, abnormal accumulation of PCs in BM, and impaired humoral immunity [3–5]. Similarly, the CXCL12CXCR4 axis can be critical for the PC neoplasm multiple myeloma. This axis regulates cell survival, cell migration, and drug resistance [55]. Our previous report showed that CD99 expression is correlated with the survival of myeloma patients [56]. The regulatory role of CD99 on CXCL12-CXCR4 axis in myeloma is remained for future study. Overall, we suggest that CD99 is a regulator of PC chemotactic migration. Investigation of the regulatory mechanism underlying PC migration will reveal therapeutic targets for regulating humoral immunity. Conflict of interest The authors declare that they have no competing interests to disclose. Acknowledgements

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This study was supported by the National Research Foundation of Korea, an MRC grant (no. 2008-0062286), and a grant from the Asan Institute for Life Sciences, Seoul, Korea (no. 2011-0794).

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