International Immunology Advance Access published March 5, 2015

1

INTIMM-14-0042 Revised 2

2 3

Human CD43+ B cells are closely related not only to memory B

4

cells phenotypically but also to plasmablasts developmentally in

5

healthy individuals

7

Masanori Inui,1* Saeko Hirota,1* Kumiko Hirano,1 Hiroshi Fujii,2 Akiko Sugahara–

8

Tobinai,1 Tomonori Ishii,2 Hideo Harigae2 and Toshiyuki Takai1

9 10

1

11

Tohoku University, Sendai 980-8575, Japan

12

2

13

Medicine, Sendai 980-8574, Japan

14

*

Department of Experimental Immunology, Institute of Development, Aging and Cancer,

Department of Hematology and Rheumatology, Tohoku University Graduate School of

These authors contributed equally to this work

15 16

Correspondence to: T. Takai, Department of Experimental Immunology, Institute of

17

Development, Aging and Cancer, Tohoku University, 4-1 Seiryo, Sendai 980-8575, Japan; E-

18

mail address: [email protected]; Tel. (81) 22-717-8501; FAX (81) 22-717-8505.

19 20

Running title: CD43+ B cells relate to memory B and plasmablasts

21 22

Total number of pages and figures: 27 pages and 6 figures and 1 supplementary figure

23 24

Keywords: natural antibody, autoantibody, ILT3/LILRB4, plasmablast/plasmacell 1

© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: [email protected]

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

6

25 26

Abstract

27 CD20+CD27+CD43+ B (CD43+ B) cells have been newly defined among peripheral blood

29

mononuclear cells (PBMCs) and proposed to be human B1 cells. However, it is controversial

30

as to whether they are orthologs of murine B1 cells and how they are related to other B cell

31

populations, particularly CD20+CD27+CD43− memory B cells and CD20lowCD27highCD43high

32

plasmablasts. Our objective is to identify phenotypically the position of CD43+ B cells among

33

peripheral B-lineage cell compartments in healthy donors, with reference to B cell subsets

34

from patients with systemic lupus erythematosus (SLE). We found that CD43+ B cells among

35

PBMCs from healthy subjects were indistinguishable phenotypically from memory B cells in

36

terms of surface markers, and spontaneous in vitro Ig and IL-10 secretion capability, but quite

37

different from plasmablasts. However, a moderate correlation was found in the frequency of

38

CD43+ B cells with that of plasmablasts in healthy donors but not in SLE patients. An in vitro

39

differentiation experiment indicated that CD43+ B cells give rise to plasmablasts more

40

efficiently than do memory B cells, suggesting that they are more closely related to

41

plasmablasts developmentally than are memory B cells, which is also supported by

42

quantitative PCR analysis of mRNA expression of B-cell and plasma-cell signature genes.

43

Thus, we conclude that, in healthy individuals, CD43+ B cells are closely related not only to

44

memory B cells phenotypically but also to plasmablasts developmentally, although the

45

developmental origin of CD43+ B cells is not necessarily the same as that of plasmablasts.

46 47

Introduction

48

Among human peripheral blood mononuclear cells (PBMCs), three distinct B cell subsets can 2

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

28

be identified on flow cytometry, i.e., naïve B, memory B, and plasmablasts, which have been

50

studied extensively in health and disease (1, 2). Deregulated B cells and antibody-forming

51

plasma cells are particularly important targets for treatment of several autoimmune diseases

52

such as systemic lupus erythematosus (SLE), because the therapeutic effect on SLE of

53

rituximab, a CD20-targeting chimeric mAb, is considered to comprise elimination of

54

activated B cells and auto-antibody-producing cells (1–6). In order to develop more efficient

55

therapeutic strategies for autoimmune diseases, it is important to elucidate the mechanism

56

underlying deregulation of B cells and plasma cells, and to find specific target molecules that

57

can modulate B cell activities to prevent autoantibody production.

58 59

In this context, the recently newly identified CD20+CD27+CD43+ B (CD43+ B) cells (or

60

proposed human B1 cells) among PBMCs, which may correspond to murine B1 cells (7, 8),

61

have attracted much attention as to how this potentially novel B cell subset is related to other

62

B cell subsets and how it is correlated to autoimmune diseases like SLE, because at least in a

63

murine system, B1 cells are considered to be the earliest onset cells among B-lineage cells,

64

and not only a major source of poly-specific and weakly autoreactive IgM natural antibodies

65

(9, 10), but also a source of IgG auto-antibody producers (11, 12). Therefore, it would be

66

interesting to determine the position of CD43+ B cells in relation to other known B cell

67

subsets. The initial series of original reports on CD43+ B cells (7, 8), however, raised

68

questions as to possible contamination by monocytes and/or T cells of the

69

CD20+CD27+CD43+ cell gate, due, in part, to incomplete elimination of doublet cells and

70

CD3+ T cells (13–15), followed by responses demonstrating that the newly identified

71

population is truly a B cell subset (8, 16). More recently, CD43+ B cells were found to possess

72

a pre-plasmablast phenotype based on the results of a functional analysis and a gene

73

expression profile (17), although this is controversial (18). Therefore, it is critically important 3

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

49

to precisely define the CD43+ B cell subset and to determine its developmental position. As

75

keys to dissect this, we were particularly interested in examining possible differences in Ig-

76

secreting ability as well as in the expression profiles of characteristic B-cell regulatory

77

receptors in B cell subsets, including a unique inhibitory IgG FcR, Fc RIIB (19, 20), sialic

78

acid-binding Ig-like lectin (Siglec)-2 (or CD22), and Siglec-10 (21–23), and inhibitory

79

isoforms of Ig-like transcript/leukocyte Ig-like receptor (ILT/LILR, also known as

80

LIR/MIR/CD85) (24–27).

81 82

The aim of the present study was to determine the characters of CD43+ B cells among PBMCs

83

from healthy individuals and to clarify their relationship with other B subsets by analyzing

84

any differences in the expression profile of B cell markers including B-cell regulatory

85

receptors and B cell/plasmacell signature genes and in the Ig-secreting ability, with reference

86

to the frequencies of B cell subsets in SLE patients. We found that CD43+ B cells from

87

healthy donors show phenotypic characteristics unexpectedly close to memory B cells but not

88

those of plasmablasts, while we also found a more close developmental relationship to

89

plasmablasts than that of memory B cells to plasmablasts in an in vitro differentiation

90

experiment, as detailed in this study.

91 92

Methods

93 94

Donors and peripheral blood samples

95 96

Twenty-six healthy donors (aged 22 – 44; 18 males and 8 females) were recruited from our

97

laboratory staff and Tohoku University Hospital with written informed consent. We also

98

collected blood samples with written informed consent from outpatients (4 males aged 27 – 4

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

74

78 and 6 females aged 22 – 67) of the Tohoku University Hospital. Patients were diagnosed as

100

SLE defined with the 1997 revised American College of Rheumatology (ACR) classification

101

criteria for SLE. All patients had active disease (SLEDAI-2K > 6) and the elevated anti-DNA

102

antibody levels in sera at the time sample obtained. Three of the ten patients had lupus

103

nephritis and two had CNS symptom. Blood samples (10–40 ml blood / each time) were

104

obtained by venipuncture using Venoject II vacuum blood collector tubes (Terumo Inc.,

105

Tokyo, Japan). This study was approved by the Ethics Committee of Tohoku University

106

Graduate School of Medicine and performed in accordance with a statement on ethical

107

principles for medical research involving human subjects made in the Declaration of Helsinki.

108 109

Reagents and antibodies

110 111

Pancoll Human and Ficoll-Paque PLUS for the purification of PBMCs were obtained from

112

PAN-Biotech GmbH (Aidenbach, Germany) and GE Healthcare Bio-Sciences AB (Uppsala,

113

Sweden), respectively. FITC-labeled mouse anti-human CD43 (clone 1G10), PerCP-Cy5.5-

114

labeled mouse anti-human CD19 (SJ25C1), and V500-labeled mouse anti-human IgD (IA6-2)

115

were purchased from BD Biosciences (San Jose, CA, USA). Allophycocyanin (APC)-Cy7-

116

labeled mouse anti-human CD20 (2H7), Brilliant Violet 510-labeled mouse anti-human CD20

117

(2H7), APC-labeled mouse anti-human CD27 (O323), Pacific Blue-labeled mouse anti-human

118

CD3 (HIT3a), PerCP-Cy5.5-labeled mouse anti-human CD19 (HIB19), Brilliant Violet 421-

119

labeled mouse anti-human CD38 (HIT2), PE-Cy7-labeled mouse anti-human CD38 (HIT2),

120

PE-Cy7-labeled mouse anti-human CD69 (FN50), PE-Cy7-labeled mouse anti-human CD5

121

(UCHT2), PE-labeled mouse anti-human IgM (MHM-88), PE-labeled rat anti-human CD32

122

(FUN-2), PE-labeled mouse anti-human CD85j (ILT2/LILRB1) (GHI/75), PE-labeled rat anti-

123

human CD85d (ILT4/LILRB2) (42D1), and PE-labeled rat anti-human CD85a 5

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

99

124

(ILT5/LILRB3) (MKT5.1) were obtained from BioLegend (San Diego, CA). PE-labeled

125

mouse anti-human CD85k (ILT3/LILRB4) (ZM4.1) was obtained from eBioscience (San

126

Diego, CA).

127 128

Cell separation

129 Mononuclear cells were obtained by density gradient separation using Pancoll Human and

131

Ficoll-Paque PLUS. We added an equal volume of PBS to the fresh blood samples, layered

132

the diluted blood in 4 ml increments onto 3 ml Pancoll or Ficoll in a 15-ml centrifugation tube

133

(Corning, Corning, NY, USA), and then centrifuged it at 430 × g for 40 min at room

134

temperature. The resultant mononuclear cell layers were collected and washed twice with

135

PBS.

136 137

Flow cytometry analysis and cell sorting

138 139

The PBMCs were subjected to immunofluorescent staining, and sort-purified on a FACS

140

Canto (BD Biosciences, San Jose, CA, USA) and a FACSAria (BD Biosciences). The data

141

were analyzed with FACS Diva (BD Bioscience) and FlowJo software (Tree Star, Inc.,

142

Ashland, OR, USA). Doublet cells were removed by gating on forward scatter (FSC)-width

143

(FSC-W) vs FSC-hight (FSC-H) according to the manufacturer's instruction. Monocytes were

144

gated based on FSC aria (FSC-A) vs SSC aria (SSC-A) characteristics.

145 146

B cell culture and stimulation

147 148

For determination of IgM and IL-10 production by B cells, sort-purified B cells were 6

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

130

149

suspended in RPMI 1640 medium supplemented with 10% FCS and 1 × 10−5 M 2-

150

mercaptoethanol, and then cultured on a 96-well round-bottom plate (Greiner Bio-one, San

151

Diego, CA) for 3–5 days in the presence or absence of 0.2 M phosphothioate–CpG-B

152

oligodeoxynucleotide (CpG-ODN; In Vivogen, San Diego, CA).

153 For induction of the development of B cells into plasmablasts in vitro, sort-purified memory

155

B cells and CD43+ B cells were suspended in Iscove-modified Dulbecco medium

156

supplemented with 10% FCS and 5 × 10−5 M 2-mercaptoethanol, 2 mM glutamax (Life

157

Technologies, Carlsbad, CA), 50 g/ml transferrin, 5 g/ml insulin, seeded at 1.5 × 105

158

cells/ml on a 24-well plate, and then stimulated with 10 g/ml CpG-ODN, 50 ng/ml histidine-

159

tagged CD40L (R&D, Minneapolis, MN), 5 g/ml anti-poly-histidine mAb (R&D), 20 U/ml

160

IL-2 (Affymetrix, San Diego, CA), 50 ng/ml IL-10 (PeproTech, Rocky Hill, NJ), and 10

161

ng/ml IL-15 (PeproTech) for 4 days, as described previously (28).

162 163

ELISA

164

ELISAs were performed to measure total IgM, IL-6, and IL-10 using Human IgM ELISA

165

Quantitation Set (Bethyl Lab, Inc., Montgomery, TX), Human IL-6 ELISA Ready-SET-Go!

166

(eBioscience, San Diego, CA), and Human IL-10 ELISA Ready-SET-Go! (eBioscience). All

167

ELISAs were performed using 96-well half-area microplates (Greiner) according to the

168

manufacturer's directions.

169 170

ELISPOT

171

7

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

154

Ig secretion was determined by ELISPOT assaying, using MultiScreen-IP plates (Millipore,

173

Billerica, MA) coated with goat anti-human IgG-Fc coating Abs (Bethyl Lab) or human IgM

174

coating Abs (Bethyl Lab), and blocked with RPMI medium. 1 × 103 sort-purified B cells were

175

cultured in 100 l RPMI medium at 37°C for 12 h, after which the plates were treated with

176

HRP-conjugated goat anti-human IgG detection Abs or HRP-conjugated human IgM

177

detection Abs and developed with 3-amino-9-ethylcarbazole. Spots were examined under a

178

fluorescence microscope and analyzed with ImageJ (National Institute of Mental Health,

179

Bethesda, MD).

180 181

Quantitative real-time PCR analysis

182 183

Total RNA was isolated using a NucleoSpin RNA XS kit (Macherey-Nagel, Dueren,

184

Germany), and subjected to cDNA synthesis with ReverTra Ace (Toyobo, Osaka, Japan). The

185

cDNA was analyzed by quantitative PCR with SYBR GreenER qPCR SuperMix (Life

186

Technologies) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules,

187

CA). The primers were used as previously described (29): BCL6 (forward, 5′-

188

GAGCTCTGTTGATTCTTAGAACTGG-3′; reverse, 5′-GCCTTGCTTCACAGTCCAA-3′),

189

PAX5 (forward, 5′-ACGCTGACAGGGATGGTG-3′; reverse, 5′-

190

CCTCCAGGAGTCGTTGTACG-3′), PRDM1 (forward, 5′-

191

AACGTGTGGGTACGACCTTG-3′; reverse, 5′-ATTTTCATGGTCCCCTTGGT-3′), and

192

XBP1 (forward, 5′-CCGCAGCACTCAGACTACG-3′; reverse, 5′-

193

TGCCCAACAGGATATCAGACT-3′). The relative gene expression is normalized by

194

GAPDH (forward, 5′-CTCTGCTCCTCCTGTTCGAC-3′; reverse, 5′-

195

ACGACCAAATCCGTTGACTC-3′).

196 8

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

172

197

Statistical analysis

198 Statistical analysis was performed using Microsoft Excel for Mac 2011 software version

200

14.2.3 (Microsoft Corp., Seattle, WA, USA). Data are displayed when appropriate as means ±

201

SD. Data were compared for statistical differences using Student's t test with two-tailed

202

analysis. P values are shown in the relevant figures. P < 0.05 was considered as statistically

203

significant.

204 205

Results

206 207

CD43+ B cells are differently gated from plasmablasts

208 209

We first attempted to isolate CD43+ B cells from PBMCs prepared from healthy volunteers,

210

essentially according to the standard protocol described previously (Fig. 1A) (7, 8). Special

211

care was taken so as to eliminate contaminating T cells and monocytes as much as possible

212

(13–15) by initially staining CD3 and CD19 in addition to standard CD20, CD27, and CD43

213

(7, 8, 30). After isolating cells at a strict lymphocyte gate, and removing doublets and CD3+

214

cells, we gated CD19+ cells as total B cells (Fig. 1A, lower left), and then identified a CD43+

215

B cell compartment neighboring the CD27−CD43−, CD27+CD43−, and CD27highCD43high cell

216

compartments, containing naive B, memory B, and plasmablasts, respectively (Fig. 1A, lower

217

middle). At this step we found that the plasmablasts defined as CD43high most precisely

218

corresponded to the conventional CD19lowCD27high plasmablasts or those on CD27highCD38++

219

gating (31; HF, TI and HH, unpublished observation).

220 221

Because the CD43+ B cell gate is adjacent to that of plasmablasts, we examined for possible 9

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

199

contamination by plasmablasts of the CD43+ B cell compartment by analyzing the expression

223

of CD38, which is highly expressed on plasmablasts (2, 32). The flow cytometric profiles of

224

CD38 expression in both the IgD− and IgD+ CD43+ B cells (see Fig. 1C) were low and

225

comparable to those in naïve and memory B cells (Fig. 1B, left). A direct comparison of the

226

CD38 expression levels between CD43+ B cells and plasmablasts by flow cytometry also

227

indicated that the expression level of CD38 on CD43+ B cells was much lower than that in

228

plasmablasts (Fig. 1B, right). These results indicate that contamination by plasmablasts, if

229

any, of the CD27+CD43+ fraction was not substantial.

230 231

Since the CD43+ B cell gate is also adjacent to the naïve and memory B cell gates, we

232

examined for possible differences in the CD43+ B cells by analyzing the expression of other B

233

cell markers, i.e., IgM, IgD and CD5. As shown in Fig. 1C, CD43+ B cells comprised

234

IgD+IgMhighCD5+ and IgDlow/−IgM−CD5− populations, whose profiles were similar to those of

235

memory B cells but different from the mostly uniform phenotype of naïve B cells exhibiting

236

IgM+IgD+CD5±. The CD5 expression levels were roughly comparable between memory B

237

and CD43+ B cells, albeit the CD5+IgD+ population was somewhat larger among CD43+ B

238

cells than memory B cells (Fig. 1C–E).

239 240

Distinguishable profiles of inhibitory receptor expression in CD43+ B cells and plasmablasts

241

but not memory B cells

242 243

The defined CD43+ B cell gate enabled us to precisely compare cell-surface regulatory

244

molecules on individual B cell subsets. Thus, we next tried to find any differences in the

245

expression profiles of regulatory receptors including inhibitory isoforms of ILT/LILR, whose

246

murine homolog is Paired Ig-like receptor (Pir)B (33, 34), a negative regulator of auto-Ab

247

production (35). In contrast to monocytes expressing the four inhibitory-type ILT/LILR

10

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

222

248

isoforms, ILT2–5/LILRB1–B4, it is believed that B cells preferentially express only

249

ILT2/LILRB1, a close homolog of murine PirB (24, 25, 36, 37). Consistent with this notion,

250

among B cells we detected only ILT2/LILRB1 on naïve, memory, and CD43+ B cells at

251

comparable levels (Fig. 2). Unexpectedly, we found that ILT3/LILRB4 expression was strong

252

on plasmablasts but not on any other B cell subsets including CD43+ B cells (Fig. 2).

253 We also found strong expression of CD32 (Fc RIIB), Siglec-10 and Siglec-2 on CD43+ B

255

cells (Fig. 2), suggesting that these receptors could be physiologically important for the

256

functional regulation of this type of B cells. Interestingly, while the Fc RIIB level on CD43+

257

B cells was not significantly different from those on naïve B cells and plasmablasts, Siglec-10

258

and -2 had almost totally disappeared on plasmablasts, whereas CD43+ B cells carried them at

259

comparable levels to those on memory B cells (Fig. 2). Comparable expression of Siglec-2

260

among the CD43+ B cells, and naïve and memory B cells has also been observed (7, 16).

261

Thus, while ILT3/LILRB4, and Siglec-10 and -2 would be useful markers for distinguishing

262

CD43+ B cells from CD27highCD43high plasmablasts, we failed to find any distinguishable

263

profiles of inhibitory receptor expression for CD43+ B cells and memory B cells.

264 265

Minimal IgM and IL-10 secretion by CD43+ B cells

266 267

Because CD43+ B cells and memory B cells were indistinguishable in the profiles of their

268

expression of IgD, CD5, ILT2/LILRB1, Siglec-10, Siglec-2, and Fc RII, we next examined if

269

spontaneous secretion of Igs or cytokines differs between CD43+ B cells and memory B cells.

270

To this end, CD43+ B, memory B and naïve B cells were sort-purified from PBMCs, and then

271

cultured for 3 or 5 days in vitro in the absence or presence of a TLR9 ligand, CpG-ODN.

272

Spontaneous secretion of IgM and IL-10 (Fig. 3A) by CD43+ B cells in the absence of the 11

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

254

TLR9 stimulator was only marginal or under the detection limit in two to three independent

274

experiments. In the presence of 0.2 M CpG-ODN, the IgM and IL-10 secretion by CD43+ B

275

cells was occasionally higher than that by memory or naïve B cells, but it was not the same

276

for every specimen tested. To confirm the poor secretion of Ig by the CD43+ B cell subset, we

277

measured the frequencies of IgM- and IgG-secreting cells by ELISPOT among CD43+ B cells

278

and in the plasmablast fraction, as a positive control. As shown in Fig. 3B, the plasmablast

279

subset prepared from three different individuals was found to be consistently rich in IgM- and

280

IgG-secreting cells, but these frequencies in CD43+ B cells were markedly low. The naïve and

281

memory B cell fractions also did not contain notable numbers of Ig-secreting cells. We

282

concluded that, like memory and naïve B cells, CD43+ B cells are not active secretors of Igs

283

and IL-10 in vitro in the absence of a stimulator. Based on the results shown in Figs. 1–3, we

284

considered at this stage that this B cell population might be more pertinent to be termed

285

CD43+ B cells with phenotypic markers very similar to memory B cells but not human B1

286

cells.

287 288

The size of CD43+ B cell compartment is not significantly altered in SLE

289 290

We were next interested in examining the CD43+ B cell compartment in PBMCs from SLE

291

patients. In SLE, the total number of PBMCs is reduced and the plasmablast compartment is

292

enlarged, while the memory B cell compartment is reduced (38, 39). We examined the size of

293

each B cell population in SLE under our flow cytometric settings. We observed a significant

294

reduction in the total number of PBMCs (Fig. 4A), and a reduction of the frequency of CD43−

295

memory B cells (Fig. 4B, C), and a marked increase in the plasmablast frequency in total

296

PBMCs (Fig. 4B) and in CD19+ cells (Fig. 4C) compared to in healthy subjects.

297

Unexpectedly, irrespective of these changes in SLE, the frequency of CD43+ B cells among

298

total PBMCs (Fig. 4B) as well as in CD19+ cells (Fig. 4C) was comparable in SLE and

12

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

273

299

healthy subjects. These results indicated that, in SLE, the size of CD43+ B cell subset is not

300

significantly changed regardless of a reduction of CD43− memory B cells or an increase of

301

plasmablasts.

302 303

A moderate correlation in the frequencies of CD43+ B cells and plasmablasts in healthy

304

subjects

306

We examined the frequency correlations between B cell compartments in healthy subjects as

307

well as in SLE patients (Fig. 5 and Supplementary Fig. S1). Obviously, care should be taken

308

when we interpret the positive or negative correlation in SLE subjects, because the donor ages

309

varied particularly in SLE patients, and the aged patients tended to have less CD43+ B cells

310

than the younger patients (Supplementary Fig. S1A). We observed, however, that the

311

correlation profiles in SLE patients did not change markedly when we omitted the plots of the

312

three patients aged over 60 (data not shown). In healthy subjects, while we did not observe

313

remarkable correlations of the frequencies of CD43+ B cells to those of total CD19+ B (Fig.

314

5A), naïve B (Fig. 5B), and memory B cells (Fig. 5C), we noted a moderate correlation

315

between the frequencies of CD43+ B cells and plasmablasts (Fig. 5D, left). In SLE,

316

interestingly, this correlation was not clear (Fig. 5D, right), but instead a correlation was

317

observed between the CD43+ B cells and the memory B cells (Fig. 5C, right). Thus, it was

318

suggested that CD43+ B cells are related to plasmablasts in healthy subjects, although CD43+

319

B cells in healthy subjects were shown to be quite similar to memory B cells phenotypically

320

and different from plasmablasts, as described above.

321 322

CD43+ B cells develop into plasmablast-like cells more efficiently than memory B cells upon

323

stimulation in vitro 13

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

305

324 To determine if the possible relation between CD43+ B cells and plasmablasts is the

326

developmental proximity between them, we sort-purified CD43+ B cells and CD43− memory

327

B cells from PBMCs of healthy subjects (Fig. 6A), and incubated them for 4 days in vitro in

328

the presence of CpG-ODN, CD40L, IL-2, IL-10 and IL-15 (28). Under this culture condition,

329

the recoveries of viable cells at day 4 were comparable between the cultures of CD43+ B cells

330

and memory B cells (Fig. 6B). We found that CD43+ B cells showed a more efficient

331

development into CD20lowCD27highCD43high plasmablast-like cells than memory B cells did

332

(Fig. 6C). CD43+ B cells were developed from the CD43− memory B cell cultures, but the

333

expression level of CD43 were lower than the CD43+ B cell cultures (Fig. 6D). Also,

334

LILRB4+ cells were developed from both CD43− memory B and CD43+ B cell cultures but

335

the frequencies of LILRB4+ cells as well as the expression levels of LILRB4 were higher in

336

CD43+ B cell cultures (Fig. 6C, D). Expression levels of CD20 and CD38 were significantly

337

lower and higher in CD43+ B cell cultures, respectively, than CD43− B cell cultures (Fig. 6E).

338

At day 4, frequencies of LILRB4+ cells were consistently higher in CD43+ B cell cultures

339

than CD43− memory B cell cultures (Fig. 6E). LILRB4+ cells at day 4 were largely

340

CD20lowCD27highCD43high, indicating that they are plasmablasts as judged by the markers (Fig.

341

6C, R2 gated cells). These results suggest that CD43+ B cells are more closely related to

342

plasmablasts developmentally than are memory B cells.

343 344

We also determined an expression profile of B cell and plasma cell signature genes in CD43+

345

B cells from healthy donors to confirm a possible developmental proximity of CD43+ B cells

346

to plasmablasts. Quantitative PCR analysis of isolated CD43+ B cells for their gene

347

expression of BCL6 and PAX5 as B-cell signatures and PRDM1 (or BLIMP1) and XBP1 as

348

plasmablast/plasmacell signatures revealed that CD43+ B cells express BCL6 and PAX5 14

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

325

mRNAs at lower levels than memory B cells (Fig. 6F). We found that CD43+ B cells also

350

express PRDM1 mRNA at a significantly higher level than memory B cells but less

351

abundantly than plasmablasts, while their XBP1 expression is significantly lower than

352

plasmablasts as those of naïve and memory B cells. PRDM1 and XBP1 expression levels in

353

CD43+ B cells after 4-day culture in the presence of stimulators and cytokines described

354

above were significantly higher than those of memory B cells (Fig. 6F). These results support

355

our notion that CD43+ B cells are more closely related to plasmablasts developmentally than

356

are memory B cells.

357 358

Discussion

359 360

In this study, we aimed at characterizing a discrete population of CD43+ B cells (or proposed

361

human B1 cells) identified recently among human PBMCs, and wanted to determine the

362

position of the cells among other B cell subsets based on their phenotypes such as the

363

expression of inhibitory receptors including several isoforms of ILT/LILR and Ig and

364

cytokine secretion. We identified a small population of CD3−CD19+CD20+CD27+CD43+ B

365

cells with nearly identical inhibitory receptor profiles to those of memory B cells and that also

366

showed inactive Ig and IL-10 secretion, and thus we suggest that this B cell population is

367

more pertinent to be called as CD43+ B cells with phenotypic markers similar to memory B

368

cells rather than human B1 cells. We also additionally found, however, a developmental

369

proximity of CD43+ B cells to plasmablasts in an in vitro development experiment.

370 371

CD43+ B cells were initially proposed to be human B1 cells, which could be a source of auto-

372

Abs as to health and disease like those in mice (7). The initial series of original reports on the

373

proposed B1 cells (7, 8), however, raised questions as to possible contamination by 15

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

349

374

monocytes and/or T cells of the CD20+CD27+CD43+ cell gate, due, in part, to incomplete

375

elimination of doublet cells and CD3+ T cells (13–15), followed by responses demonstrating

376

that the newly identified B cell population is truly a B cell subset (8, 16). Using a more

377

stringent gating strategy to eliminate T cells and monocytes, we also identified a small but

378

discrete B cell lineage population, as judged from its exclusive ILT2/LILRB1 expression (Fig.

379

2) in addition to the CD3−CD19+CD20+ phenotype (Fig. 1).

381

The proposed B1 subset was shown to be sub-dividable into two discrete populations (40),

382

one being CD11b+ B1 or "orchestrator B1 cells", which can stimulate allogeneic T cell

383

proliferation and also can suppress CD3-activated T cells via IL-10 secreted by the CD11b+

384

B1 cells, and the other being CD11b− B1 cells or "secretor B1 cells", which spontaneously

385

secrete IgM Abs. Later, other groups (41, 42) tried to find some discrete characters of the

386

proposed B1 cells and their relation to common variable immunodeficiencies. Each group

387

identified a proposed B1 cell population, but some of the characters initially reported (7) were

388

not confirmed (41, 42). We failed to observe spontaneous and active production of Ig or IL-10

389

by CD43+ B cells in vitro (Fig. 3).

390 391

Concerning the sub-components of the proposed B1 cells, it has been documented that the B1

392

cell fraction comprises IgG+ or IgA+ cells in addition to IgD+ cells, arguing that this fraction

393

may be contaminated by activated CD38+ plasma cells or their precursors (13, 14). Analysis

394

of our CD43+ B cells indicated that they could be subdivided into IgD+IgM+CD5+ and

395

IgD−IgM−CD5− populations, the IgD− fraction being dominant, and these two component

396

profiles are close to that of the CD43− memory B cell subset, which is also composed of

397

IgD+IgM+CD5+ and IgD−IgM−CD5− cells, the latter also being dominant (Fig. 1). The

398

CD38low profile of CD43+ B cells (Fig. 1) indicated successful elimination of substantial 16

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

380

399

contamination by CD38+ plasmablasts of the subset.

400 It has been recently argued that the proposed human B1 cells exhibit a pre-plasmablast

402

phenotype (17). They showed that CD20+CD27+CD43+ B cells could develop from memory

403

B cells upon stimulation with R-848, a ligand for TLR7 and TLR8, and IL-2 for 5 days. They

404

also showed that the proposed B1 cells could become a substantial number of CD20−

405

plasmablasts and a small number of plasma cells upon stimulation with R-848, IL-2, IL-10,

406

IL-15 and IL-6 for 5 days. Microarray analysis of high-expression gene profiles in the

407

proposed B1 cells, memory B cells, and plasmablasts also suggested that the proposed B1

408

cells were closer to plasmablasts than memory B cells. Based on these data, they concluded

409

that the proposed B1 cells possess pre-plasmablast phenotypes that are inconsistent with

410

murine B1 cells, although controversy still exists (18, 43, 44). In our present study, the data

411

on marker and Ig/cytokine secretion profiles suggest that CD43+ B cells are a B cell subset

412

with phenotypes nearly identical to those of memory B cells. However, we noted a moderate

413

correlation in the frequencies of CD43+ B cells and plasmablasts in healthy subjects (Fig. 5D),

414

which prompted us to examine the developmental proximity of CD43+ B cells to plasmablasts

415

by means of quantitative PCR analysis of signature genes and in vitro induction of

416

plasmablasts from CD43+ B cells by culturing them with a TLR9 ligand, CD40L and

417

cytokines (Fig. 6). While the expression profile of B cell-specific genes in CD43+ B cells was

418

intermediate of memory B cells and plasmablasts, we found that, as Covens et al. (17)

419

reported, CD20lowCD27highCD43high plasmablasts were induced from CD43+ B cells. In

420

addition, we also found that the induction was more efficient for CD43+ B cells than CD43−

421

memory B cells, suggesting that CD43+ B cells are more closely related to plasmablasts than

422

are memory B cells. We consider, however, that the origin of CD43+ B cells is not necessarily

423

CD43− memory B cells and that plasmablasts are not necessarily generated from CD43+ B 17

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

401

424

cells in vivo, because we did not examine their precise developmental fate nor did verify the

425

identity of CD43+ B cells generated in the culture with those cells in PBMCs but we only

426

examined developmental markers and expression of several signature genes. Covens et al.

427

(17) pointed out that CD43+ B cells represent activated CD27+ B cells that can be derived

428

from different B cell subsets.

429 CD43+ B cells are clearly distinguishable from plasmablasts phenotypically in their positive

431

Siglec-10 and Siglec-2 but negative ILT3/LILRB4 expression. Exclusive expression of

432

ILT2/LILRB1 but not other inhibitory ILT/LILR isoforms including ILT4/LILRB2,

433

ILT5/LILRB3 and ILT3/LILRB4 on the CD43+ B cells as well as the naïve and memory B

434

cell subsets was anticipated, because ILT2/LILRB1 is the sole ILT/LILR isoform expressed

435

on human B cells (27, 37, 45). One of the interesting findings in our present study was that

436

plasmablasts exhibited striking induction of ILT3/LILRB4 in addition to the B-lineage cell

437

signature, ILT2/LILRB1 expression (Fig. 2), although a preceding microarray-based study

438

revealed significant overexpression of ILT3/LILRB4 mRNA in polyclonal plasmablastic cells

439

compared to in mature plasma cells (46). We found that ILT3/LILRB4 is a useful marker not

440

only for plasmablasts in PBMCs but also for plasmablasts in culture, as is the case for CD38,

441

as demonstrated in an in vitro differentiation experiment shown in Fig. 6. ILT2/LILRB1 is

442

expressed on B cells, a subset of T cells, NK cells, monocytes and macrophages, and dendritic

443

cells, and is one of two human orthologs of murine PirB (25, 33, 34, 45, 47). Although

444

ILT4/LILRB2 is another ortholog of PirB in terms of structural and functional similarities, it

445

is not expressed on human B cells but on other cells in which ILT2/LILRB1 co-exists. The

446

ligands for ILT2/LILRB1 are multiple, including HLA-A, B, C, E and F (45),

447

cytomegalovirus protein UL18 (26), neurite outgrowth inhibitor protein or Nogo, myelin-

448

associated glycoprotein or MAG, and oligodendrocyte myelin glycoprotein or OMgp (48). 18

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

430

ILT4/LILRB2 ligands include also HLA class I molecules, and three kinds of neuronal myelin

450

proteins, Nogo/MAG/OMgp, and several angiopoietin-like proteins (Angptls), a family of

451

seven secreted glycoproteins (49), but ILT4/LILRB2 does not bind UL18. ILT3/LILRB4 is

452

found on monocytes/macrophages and dendritic cells, but the ligand for ILT3/LILRB4 is

453

currently not known. There has been no extensive study on the physiological role of

454

ILT3/LILRB4, except for ones showing a modulatory effect of this receptor in making

455

dendritic cells tolerogenic (50, 51). Our finding of surface ILT3/LILRB4 expression on

456

plasmablasts suggests that this receptor with cytoplasmic signal-inhibiting motifs plays a

457

modulatory role in some developmental stages of plasmablasts or plasma cells, which will

458

home to peripheral tissues and bone marrow where they constitutively produce Abs.

459 460

In conclusion, CD43+ B cells among PBMCs possess very similar phenotypic characteristics

461

to those of memory B cells, while they exhibit developmental proximity to plasmablasts more

462

than memory B cells do. We suggest that CD43+ B cells are a potent precursor population en

463

route to plasmablasts or an activated B cell subset or both in healthy individuals. Whether the

464

CD43+ B cell subset is always at an intermediate stage of the development from memory B

465

cells to plasmablasts, or a discrete, independent population with a potential to become

466

plasmablasts in health and disease remains an open question.

467 468

Funding

469 470

Core Research for Evolutional Science and Technology Program of the Japan Science and

471

Technology Agency (to T.T.), Grant-in-Aid from the Ministry of Education, Culture, Sports,

472

Science and Technology of Japan (to T.T. and M.I.), and a grant from the Global Center of

473

Excellence Program for Innovative Therapeutic Development Towards the Conquest of Signal 19

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

449

474

Transduction Diseases with Network Medicine (to T.T.).

475 476

Acknowledgments

477 478

We thank Nicholas Halewood for the editorial assistance.

479 Conflict of interest statement: The authors declared no conflict of interests.

481 482

References

483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514

1

Jackson, S. M., Wilson, P. C., James, J. A. and Capra J. D. 2008. Human B cell subsets. Adv. Immunol. 98:151.

2

Dörner, T., Giesecke, C. and Lipsky, P. E. 2011. Mechanisms of B cell autoimmunity in SLE. Arthritis Res. Ther. 13:243.

3

Furtado, J. and Isenberg, D. A. 2013. B cell elimination in systemic lupus erythematosus. Clin. Immunol. 146:90.

4

Vital, E. M., Dass, S., Buch, M. H., Henshaw, K., Pease, C. T., Martin, M. F., Ponchel, F., Rawstron, A. C. and Emery, P. 2011. B cell biomarkers of rituximab responses in systemic lupus erythematosus. Arthritis Rheum. 63:3038.

5

Stohl, W., Hiepe, F., Latinis, K. M., Thomas, M., Scheinberg, M. A., Clarke, A., Aranow, C., Wellborne, F. R., Abud-Mendoza, C., Hough, D. R., Pineda, L., Migone, T. S., Zhong, Z. J., Freimuth, W. W., Chatham, W. W.; BLISS-52 Study Group; BLISS-76 Study Group. 2012. Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with systemic lupus erythematosus. Arthritis Rheum. 64:2328.

6

Rovin, B. H., Furie, R., Latinis, K., Fervenza, F. C., Sanchez-Guerrero, J., Maciuca, R., Zhang, D., Garg, J. P., Brunetta, P., Appel, G.; LUNAR Investigator Group. 2012. Efficacy and safety of rituximab in patients with active proliferative lupus nephritis: the Lupus Nephritis Assessment with Rituximab study. Arthritis Rheum. 64:1215.

7

Griffin, D. O., Holodick, N. E. and Rothstein, T. L. 2011. Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70−. J. Exp. Med. 208:67.

8

Griffin, D. O. and Rothstein, T. L. 2011. A small CD11b+ human B1 cell subpopulation stimulates T cells and is expanded in lupus. J. Exp. Med. 208:2591. 20

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

480

550 551 552 553 554 555 556 557 558 559 560 561 562

9

Avrameas, S., Ternynck, T., Tsonis, I. A. and Lymberi, P. 2007. Naturally occurring Bcell autoreactivity: a critical overview. J. Autoimmun. 29:213.

10

Ehrenstein, M. R. and Notley, C. A. 2010. The importance of natural IgM: scavenger, protector and regulator. Nat. Rev. Immunol. 10:778.

11

Baumgarth, N., Tung J. W. and Herzenberg L. A. 2005. B-1 B cells: devel- opment, selection, natural autoantibody and leukemia. Springer Semin. Immunopathol. 26:347.

12

Carroll, M. C. and Holers V. M. 2005. Innate autoimmunity. Adv. Immunol. 86:137.

13

Descatoire, M., Weill, J. C., Reynaud, C. A. and Weller, S. 2011. A human equivalent of mouse B-1 cells? J. Exp. Med. 208:2563.

14

Perez-Andres, M., Grosserichter-Wagener, C., Teodosio, C., van Dongen, J. J., Orfao, A. and van Zelm, M. C. 2011. The nature of circulating CD27+CD43+ B cells. J. Exp. Med. 208:2565.

15

Reynaud, C. A. and Weill, J. C. 2012. Gene profiling of CD11b+ and CD11b− B1 cell subsets reveals potential cell sorting artifacts. J. Exp. Med. 209:433.

16

Griffin, D. O., Quach, T., Batliwalla, F., Andreopoulos, D., Holodick, N. E. and Rothstein, T. L. 2012. Human CD11b+ B1 cells are not monocytes: A reply to "Gene profiling of CD11b+ and CD11b− B1 cell subsets reveals potential cell sorting artifacts". J. Exp. Med. 209:434.

17

Covens, K., Verbinnen, B., Geukens, N., Meyts, I., Schuit, F., Van Lommel, L., Jacquemin, M. and Bossuyt, X. 2013. Characterization of proposed human B-1 cells reveals pre-plasmablast phenotype. Blood 121:5176.

18

Li, W., Batliwalla, F. and Rothstein, T. L. 2013. Human B-1 cells are not preplasmablasts: analysis of microarray data and other issues. Blood 122:3691.

19

Nimmerjahn, F. and Ravetch, J. V. 2008. Fc receptors as regulators of immune responses. Nat. Rev. Imunol. 8:34.

20

Takai, T., Ono, M., Hikida, M., Ohmori, H. and Ravetch, J. V. 1996. Augmented humoral and anaphylactic responses in Fc RII-deficient mice. Nature 379:346.

21

Nitschke, L., Carsetti, R., Ocker, B., Köhler, G. and Lamers, M. C. 1997. CD22 is a negative regulator of B-cell receptor signalling. Current Biology 7:133.

22

Nitschke, L. 2009. CD22 and Siglec-G: B-cell inhibitory receptors with distinct function. Immunol. Rev. 230:128.

23

Jellusova, J., Wellmann, U., Amann, K., Winkler, T. H. and Nitschke, L. 2010. CD22 × Siglec-G double-deficient mice have massively increased B1 cell numbers and develop systemic autoimmunity. J. Immunol. 184:3618.

21

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549

24

Cella, M., Döhring, C., Samaridis, J., Dessing, M., Brockhaus, M., Lanzavecchia, A. and Colonna, M. 1997. A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing. J. Exp. Med. 185:1743.

25

Colonna, M., Navarro, F., Bellon, T., Llano, M., Garcia, P., Samaridis, J., Cella, M. and Lopez-Botet, M. 1997. A common inhibitory receptor for major histocompatibility complex class I molecules on human lymphoid and myelomonocytic cells. J. Exp. Med. 186:1809.

26

Cosman, D., Fanger, N., Borges, L., Kubin, M., Chin, W., Peterson, L. and Hsu, M. L. 1997. A novelimmunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7:273.

27

Brown, D., Trowsdale, J. and Allen, R. 2004. The LILR family: modulators of innate and adaptive immune pathways in health and disease. Tissue Antigens 64:215.

28

Jourdan, M., Caraux, A., De Vos, J., Fiol, G., Larroque, M., Cognot, C., Bret, C., Duperray, C., Hose, D. and Klein, B. 2009. An in vitro model of differentiation of memory B cells into plasmablasts and plasma cells including detailed phenotypic and molecular characterization. Blood 114:5173.

29

Avery, D. T., Deenick, E. K., Ma, C. S., Suryani, S., Simpson, N., Chew, G. Y., Chan, T. D., Palendira, U., Bustamante, J., Boisson-Dupuis, S., Choo, S., Bleasel, K. E., Peake, J., King, C., French, M. A., Engelhard, D., Al-Hajjar, S., Al-Muhsen, S., Magdorf, K., Roesler, J., Arkwright, P. D., Hissaria, P., Riminton, D. S., Wong, M., Brink, R., Fulcher, D. A., Casanova, J. L., Cook, M. C. and Tangye, S. G. 2010. B cell-intrinsic signaling through IL-21 receptor and STAT3 is required for establishing long-lived antibody responses in humans. J. Exp. Med. 207:155.

30

Griffin, D. O. and Rothstein, T. L. 2012. Human B1 cell frequency: isolation and analysis of human B1 cells. Front. Immunol. 3:122.

31

Odendahl, M., Jacobi, A., Hansen, A., Feist, E., Hiepe, F., Burmester, G. R., Lipsky, P. E., Radbruch, A. and Dörner, T. 2000. Disturbed peripheral B lymphocyte homeostasis in systemic lupus erythematosus. J. Immunol. 165:5970.

32

Dörner, T., Jacobi, A. M., Lee, J. and Lipsky, P. E. 2010. Abnormalities of B cell subsets in patients with systemic lupus erythematosus. J. Immunol. Methods. 363:187.

33

Hayami, K., Fukuta, D., Nishikawa, Y., Yamashita, Y., Inui, M., Ohyama, Y., Hikida, M., Ohmori, H. and Takai, T. 1997. Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors. J. Bio. Chem. 272:7320.

34

Kubagawa, H., Burrows, P. D. and Cooper, M. D. 1997. A novel pair of immunoglobulin-like receptors expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA. 94:5261.

35

Kubo, T., Uchida, Y., Watanabe, Y., Abe, M., Nakamura, A., Ono, M., Akira, S. and Takai, T. 2009. Augmented TLR9-induced Btk activation in PIR-B-deficient B-1 cells provokes excessive autoantibody production and autoimmunity. J. Exp. Med. 206:1971. 22

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612

36

Shiroishi, M., Kuroki, K., Rasubala, L., Tsumoto, K., Kumagai, I., Kurimoto, E., Kato, K., Kohda, D. and Maenaka, K. 2006. Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d). Proc. Natl. Acad. Sci. USA. 103:16412.

37

Kuroki, K., Tsuchiya, N., Shiroishi, M., Rasubala, L., Yamashita, Y., Matsuta, K., Fukazawa, T., Kusaoi, M., Murakami, Y., Takiguchi, M., Juji, T., Hashimoto, H., Kohda, D., Maenaka, K. and Tokunaga, K. 2005. Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis. Hum. Mol. Genet. 14:2469.

38

Jackson S. M., Wilson P. C., James J. A. and Capra J. D. 2008. Human B cell subsets. Adv. Immunol. 98:151.

39

Dörner T., Jacobi A. M., Lee J. and Lipsky P. E. 2011. Abnormalities of B cell subsets in patients with systemic lupus erythematosus. J. Immunol. Methods 363:187.

40

Griffin, D. O. and Rothstein, T. L. 2012. Human "orchestrator" CD11b+ B1 cells spontaneously secrete interleukin-10 and regulate T-cell activity. Mol. Med. 18:1003.

41

Suchanek, O., Sadler, E. A., Patel, S. Y. and Ferry, B. L. 2012. Immunophenotyping of putative human B1 B cells in healthy controls and common variable immunodeficiency (CVID) patients. Clin. Exp. Immunol. 170:333.

42

Kraljevic, K., Wong, S. and Fulcher, D. 2012. Circulating phenotypic B-1 cells are decreased in common variable Immunodeficiency and Correlate with IgM levels. Clin. Exp. Immunol. 171:278.

43

Covens, K., Verbinnen, B., Jacquemin, M. and Bossuyt, X. 2013. Response: Extended analysis of microarray data does not contradict preplasmablast phenotype of human CD20+CD27+CD43+ cells. Blood 122:3693.

44

Mabbott, N. A. and Gray, D. 2013. Identification of co-expressed gene signatures in mouse B1, marginal zone and B2 B-cell populations. Immunology 141:79.

45

Cella, M., Nakajima, H., Facchetti, F., Hoffmann, T. and Colonna, M. 2000. ILT receptors at the interface between lymphoid and myeloid cells. Curr. Top. Microbiol. Immunol. 251:161.

46

Tarte, K., Zhan, F., De Vos, J., Klein, B. and Shaughnessy Jr., J. 2003. Gene expression profiling of plasma cells and plasmablasts: toward a better understanding of the late stages of B-cell differentiation. Blood 102:592.

47

Borges, L., Hsu, M. L., Fanger, N., Kubin, M. and Cosman, D. 1997. A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules. J. Immunol. 159:5192.

23

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660

48

Atwal, J. K., Pinkston-Gosse, J., Syken, J., Stawicki, S., Wu, Y., Shatz, C. and TessierLavigne, M. 2008. PirB is a functional receptor for myelin inhibitors of axonal regeneration. Science 322:967.

49

Zheng, J., Umikawa, M., Cui, C., Li, J., Chen, X., Zhang, C., Huynh, H., Kang, X., Silvany, R., Wan, X., Ye, J., Cantó, A. P., Chen, S. H., Wang, H. Y., Ward, E. S. and Zhang, C. C. 2012. Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature 485:656.

50

Chang C. C., Ciubotariu, R., Manavalan, J. S., Yuan, J., Colovai, A. I., Piazza, F., Lederman, S., Colonna, M., Cortesini, R., Dalla-Favera, R. and Suciu-Foca, N. 2002. Tolerization of dendritic cells by T(S) cells: the crucial role of inhibitory receptors ILT3 and ILT4. Nat. Immunol. 3:237.

51

Brown, D. P., Jones, D. C., Anderson, K. J., Lapaque, N., Buerki, R. A., Trowsdale, J. and Allen, R. L. 2009. The inhibitory receptor LILRB4 (ILT3) modulates antigen presenting cell phenotype and, along with LILRB2 (ILT4), is upregulated in response to Salmonella infection. BMC Immunol. 10:56.

681

Figure legends

682 683

Fig. 1. Flow cytometric identification of a CD43+ B cell subset and its IgD, IgM, and CD5

684

expression.

685

(A) After gating for lymphocytes (upper left), doublet elimination by gating on FSC-W vs

686

FSC-H (upper middle), and isolation of a CD3− fraction (upper left), CD19+ cells (lower left)

687

were analyzed for CD43 and CD27 expression (lower middle). Naïve B, memory B and

688

CD43+ B cells, and plasmablasts were defined as CD27−CD43−, CD27+CD43−, CD27+CD43+,

689

and CD27+CD43high cells, respectively. The isotype control for CD43 did not stain the CD43+

690

B cell fraction (lower right). (B) Surface expression of CD38 was determined by

691

immunofluorescent staining and flow cytometry, and the expression levels are presented for

692

naïve B, IgD− or IgD+ memory B, and IgD− or IgD+ CD43+ B cells, plasmablasts, and

693

monocytes, as a positive control. Isotype control staining is shown in solid gray (left). CD38

694

expression levels are shown as means ± SD (n = 5, right). Monocytes were gated by the FSC-

695

A vs SSC-A profile. (C–E) CD43+ B cells comprise two distinct populations,

24

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680

IgD+IgMhighCD5+ and IgD−IgM−CD5−. (C) Analysis of IgD, IgM, and CD5 expression on

697

naïve B, memory B, and CD43+ B cells by flow cytometry. In memory B and CD43+ B cells,

698

the IgD+IgMhigh populations (gated as upper right squares in left column) were found to be

699

IgD+CD5+ (red dots in middle column), and CD5+IgMhigh (red dots in right column).

700

Conversely, IgD−IgM− populations (gated as lower left squares in left column) were identified

701

as IgD−CD5− (blue dots in middle column), and CD5−IgM− (blue dots in right column). (D)

702

Naïve B, memory B, and CD43+ B cells were analyzed for their IgD and CD5 expression to

703

compare the CD5+ populations. The isotype control for CD5 is shown in the left panel. (E)

704

The percentages of CD5+ cells and CD5 expression levels of IgD− and IgD+ CD43+ B cells

705

were compared to those for naïve B cells, and IgD− and IgD+ memory B cells. Data are means

706

± SD for three independent experiments. The profiles of CD43+ B cells were mostly similar to

707

those of memory B cells, but CD5+ cells were more numerous among IgD+ puB1 cells than

708

IgD+ memory B cells.

709 710

Fig. 2. CD43+ B cells express a series of inhibitory receptors characteristic of B cells.

711

Histograms (A) and graphs (B) showing the expression levels of a series of inhibitory

712

receptors on each B cell population as well as on monocytes as a control (A). In the graphs,

713

data are means ± SD for three independent experiments. While monocytes expressed all of the

714

four types of LILRB examined, both IgD− and IgD+ CD43+ B cells expressed only LILRB1 at

715

similar levels to those in other B cell populations, but were negative for LILRB2, B3, and B4

716

(A), such an expression preference for LILRB1 being a hallmark of B-lineage cells. Higher

717

expression was found for LILRB4 on plasmablasts than the other B cell subsets including

718

CD43+ B cells (B). The expression of Fc RII, Siglec-10, and CD22 (A, B) was found on both

719

the IgD− and IgD+ CD43+ B cells. *P < 0.05; ***P < 0.005; ns, not significant.

720 25

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

696

721

Fig. 3. IgM, IgG, and IL-10 production from CD43+ B cells were comparable to those from

722

naïve and memory B cells.

723

(A) Production of IgM (left) and IL-10 (right) in the absence (control) or presence of CpG M for naïve B and memory B cells, and only 0.2 M for CD43+ B cells due

725

to the limited cell number) in a culture of each B cell fraction for 5 days. Data are expressed

726

as means ± SD for three wells, except that the IgM levels for CD43+ B cells in Expt. 1 and 3

727

are expressed as means for two wells due to the limited cell numbers. In the absence of

728

stimulation, IgM production was negligible. Upon CpG-ODN stimulation, each B cell

729

population produced IgM and IL-10, but the amounts released were not markedly different

730

among the populations. (B) ELISPOT assaying indicates that the CD43+ B cell fraction does

731

not contain a significant number of IgM- or IgG-producing cells. CD43+ B cells and the other

732

B subset cells were subjected to ELISPOT assaying to detect (upper) IgM- and (lower) IgG-

733

producing cells. Sort-purified B cells were plated at 1–7 × 103 cells per well and then

734

incubated for 12 h at 37°C. The graphs are for three independent experiments. Data with

735

error bars are means ± SD for three wells, and those without error bars are from single well

736

determinations due to the limited number of sort-purified cells.

737 738

Fig. 4. Frequency of each B cell subset in total PBMCs and in CD19+ cells in healthy donors

739

and SLE patients.

740

PBMCs were prepared from healthy donors and SLE patients (A), and analyzed by flow

741

cytometry, and then the size of each B cell subset as the frequency in total PBMCs (B, means

742

± SD, n = 9 for healthy and n = 10 for SLE) and that in CD19+ cells (C, means ± SD, n = 9

743

for healthy and n = 10 for SLE) was calculated. *P < 0.05, **P < 0.01, ***P < 0.001.

744 745

Fig. 5. Correlation study on CD43+ B cell frequency as to those of total B cells, naïve B cells, 26

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

724

746

memory B cells and plasmablasts in healthy donors and SLE patients.

747

PBMCs were analyzed by flow cytometry after immunofluorescent staining and the

748

correlations between the frequencies of CD43+ B cells and total CD19+ B cells in total

749

PBMCs (A), and CD43+ B cells and naïve B cells (B), CD43+ B cells and memory B cells

750

(C), and CD43+ B cells and plasmablasts (D) in total CD19+ B cells were determined.

751 Fig. 6. CD43+ B cells develop into plasmablast-like cells more efficiently than memory B

753

cells upon stimulation in vitro.

754

CD43+ B cells and CD43− memory B cells were sort-purified from PBMCs of healthy

755

subjects (A), and incubated for 4 days in vitro in the presence of CpG-ODN, CD40L, IL-2,

756

IL-10 and IL-15. (B) Recovery of viable cells at day 4. (C) Flow cytometric analysis of the

757

cells at day 4. The CD43+ B cell culture contained more LILRB4+CD20lowCD27highCD43high

758

plasmablast-like cells than the CD43− memory B cell culture. (D) Histograms for the CD43+

759

and LILRB4+ cells at day 4. (E) Comparison of expression levels of CD20, frequencies of

760

CD38+ cells, and differences in LILRB4+ cell frequencies of CD43− memory B and CD43+ B

761

cell cultures at day 4. (F) Expression of B cell/plasmacell transcription factors in each isolated

762

B cell subset from healthy donors (Day 0) and in the CD43+ B cells and memory B cells

763

cultured for 4 days (Day 4) were determined by quantitative PCR. The relative expression of

764

transcripts was normalized to GAPDH. Data are means ± SD for three independent

765

experiments. *P < 0.05, **P < 0.01.

27

Downloaded from http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

752

A

C

FSC-A

SSC-A

SSC-A

FSC-W

Naïve B

CD3

FSC-H

Memory B

33.9

39.2

CD5

CD43+ B IgM

CD20

CD27

CD27

56.3

35000 30000

Isotype for CD5

Isotype control CD38

25000 20000 15000 10000

Naïve B

Memory B

CD43+ B

0.3

0.8

0.9

17

0.7

3.1

3.5

8.5

64.8

34.1

6.1

76

65.5

30.7

73.2

14.9

5000 0

CD5

IgD

IgD

E 20 20

CD5 expression (MFI)

Mean fluorescence intensity

% of maximum ■ Isotype control ー Monocyte ー Naïve B ー Memory B (IgD−) ー Memory B (IgD+) ー CD43+ B (IgD−) ー CD43+ B (IgD+) ー Plasmablast

IgD

D

CD38

Fluorescence intensity

47.3

CD5

B

Isotype control

CD43

CD5-positive cells (%)

CD19

IgM

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 1

15 15 10 10

55 00

Naïve B IgD− IgD+ IgD− IgD+ Memory B

CD43+ B

1500 1500 1000 1000

500 500 00

Naïve B IgD− IgD+ IgD− IgD+ Memory B CD43+ B

LILRB1

LILRB2

LILRB3

LILRB4

LILRB1

1000 800

MFI

3000

CD22

■ Isotype control ー Monocyte ー Naïve B ー Memory B (IgD−) ー Memory B (IgD+) ー CD43+ B (IgD−) ー CD43+ B (IgD+) ー Plasmablast

600

2000

400

1000

200

0

0

Fc RIIB 80000 60000

MFI

Siglec-10

LILRB4

ns

4000

Fc RII % of maximum

B ns

% of maximum

A

ns

0

*

Isotype control Inhibitory receptor

Siglec-2 (CD22)

Siglec-10 ns

1500 1000

40000 20000

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 2

500

ns

*

25000 20000 15000 10000 5000

0

0

ns ***

Expt. 3 300

250

200

150

100 50 0

0

Spot count per 2 x 103 cells plated

Spot count per 1 x 103 cells plated

Expt. 2 Spot count per 7 x 103 cells plated

IgM production (ng/ml)

500

400

300

200

100 0

Spot count per 2 x 103 cells plated

IL-10

Spot count per 1 x 103 cells plated

IgM production (ng/ml)

Expt. 1 IgM

Spot count per 1 x 103 cells plated

IgM production (ng/ml)

A

B IgM

naïve mem 43+B PB

14 12 10 8 6 4 2 0

naïve mem 43+B PB

IgG

naïve mem 43+B PB

120

100

80

60

40

20

naïve mem 43+B PB

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 3

60

50

40

30

20

10 0

naïve mem 43+B PB

100 80

60

40

20 0

naïve mem 43+B PB

A *

2.5

Frequency (%)

1.5 1 0.5 0

SLE (n = 10)

**

2

P = 0.08

1

0

SLE

Naïve B 80 60 40 20 0

Healthy

SLE

CD43+ B 25 20 15 10 5 0 -5

Healthy

SLE

Frequency in CD19+ cells (%)

Frequency in CD19+ cells (%) Frequency in CD19+ cells (%)

Frequency in total PBMCs

Healthy (n = 9)

2

Healthy

C

3

Frequency in CD19+ cells (%)

PBMC number (x106/ml)

B

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 4

Memory B P = 0.05

50 40 30 20 10 0

Healthy

SLE

Plasmablast

***

30

20

10

0

Healthy

SLE

Total B vs CD43+ B Correlation = 0.15 (n = 23)

0.4 0.2 0

0.4 0.2 0

0

5

10

15

0

Total B (%)

2

4

6

14 12 10 8 6 4 2 0

8

Correlation = −0.48 (n = 26)

20

40

60

80

Naïve B (%)

Total B (%)

C

D Healthy

SLE

Healthy Correlation = 0.76 (n = 9)

12 10 8 6 4 2 0

0

50 100 Memory B (%)

10 8

Correlation = 0.55 (n = 26)

6 4 2 0

0

12

Correlation = −0.75 (n = 9)

10 8 6 4 2 0

30

40

10 20 30 Memory B (%)

40

50

60

70

Naïve B (%)

SLE

CD43+ B vs Plasmablast Plasmablast (%)

Correlation = 0.13 (n = 26)

CD43+ B (%)

CD43+ B (%)

Memory B vs CD43+ B 14 12 10 8 6 4 2 0

SLE

Naïve B vs CD43+ B

CD43+ B (%)

0.6

Healthy

Correlation = 0.91 (n = 9)

0.6 CD43+ B (%)

CD43+ B (%)

0.8

B

SLE

CD43+ B (%)

Healthy

30 Plasmablast (%)

A

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 5

Correlation = −0.07 (n = 9)

20 10 0

0

10 CD43+ B (%)

20

0

2

4 6 8 10 12 CD43+ B (%)

B

Day 0

CD27

CD27 CD43

C

CD43+ B

CD43 memory

Cell number (x105)

CD43+ B

CD27

CD43 memory

CD19

3 2

CD43 CD43+

1 0

Day 4

Day 0

CD19

Day 4 R1 gated

R1 gated LILRB4

R2

R2

SSC

SSC

CD27

R1

CD20

CD43− memory B

SSC

Isotype control

9.2

0.5

91.1

FSC

CD19

CD43

R1 gated

R1 gated

CD20

R1 gated

R1 gated

LILRB4

LILRB4

R1 gated

R2 gated

R2 gated

CD27

A

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 6A–E

CD19

CD43

R2 gated

R2 gated

19.7

CD43

E 48 87

CD43

CD43 memory B Day 0 Day 4 CD43+ B Day 0 Day 4 211

% of maximum

% of maximum

D 3.3 6.4 10.1

LILRB4

Isotype control CD43 memory B CD43+ B

CD19

LILRB4

400 300 200 100 0

* CD38+ cells (%)

CD19

CD20 expression (MFI)

FSC

20 15 10 5 0

*

Fold increase in % of LILRB4+ cells

91.7

8

CD27

R2

CD20

R1

SSC

CD20

SSC

CD43+ B

CD27

LILRB4

3 2 1

0

CD43

*

BCL6 mRNA relative expression (x10 4) 0

Day 0

0.8

0.6

0.4

0.2

Day 4 Day 0

1.5

*

0.5 0

Day 4

**

Day 0

0.03

* *

0

Day 4

XBP1 mRNA relative expression

Memory B CD43+ B

0.02

Plasmablast

0.01

Memory B CD43+ B

Naïve B

PRDM1 mRNA relative expression

Memory B CD43+ B

*

Plasmablast

1

Memory B CD43+ B

Naïve B

PAX5 mRNA relative expression

Memory B CD43+ B

Plasmablast

Memory B CD43+ B

Naïve B

F

0.1

Day 0

Memory B CD43+ B

0.2

Plasmablast

Memory B CD43+ B

Naïve B

http://intimm.oxfordjournals.org/ at University of California, San Francisco on March 26, 2015

Figure 6F

0.3

** *

0

Day 4