gb-2008-9-1-r17 GBJ Research <p>Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling</p> Robbins H Scott SRobbins@gnf.org Walzer Thierry walzer@ciml.univ-mrs.fr Dembélé Doulaye doulaye@igbmc.u-strasbg.fr Thibault Christelle thibault@igbmc.u-strasbg.fr Defays Axel defays@ciml.univ-mrs.fr Bessou Gilles bessou@ciml.univ-mrs.fr Xu Huichun huixu@ucdavis.edu Vivier Eric vivier@ciml.univ-mrs.fr Sellars MacLean msellars@igbmc.u-strasbg.fr Pierre Philippe pierre@ciml.univ-mrs.fr Sharp R Franck frank.sharp@ucdmc.ucdavis.edu Chan Susan chan@igbmc.u-strasbg.fr Kastner Philippe kastner@igbmc.u-strasbg.fr Dalod Marc dalod@ciml.univ-mrs.fr

CIML (Centre d'Immunologie de Marseille-Luminy), Université de la Méditerranée, Parc scientifique de Luminy case 906, Marseille F-13288, France

U631, INSERM (Institut National de la Santé et de la Recherche Médicale), Parc scientifique de Luminy case 906, Marseille F-13288, France

UMR6102, CNRS (Centre National de la Recherche Scientifique), Parc scientifique de Luminy case 906, Marseille F-13288, France

Hematopoiesis and leukemogenesis in the mouse, IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), rue Laurent Fries, ILLKIRCH F-67400, France

U596, INSERM, rue Laurent Fries, ILLKIRCH F-67400, France

UMR7104, CNRS, rue Laurent Fries, ILLKIRCH F-67400, France

UM41, Université Louis Pasteur, rue Laurent Fries, Strasbourg F-67400, France

The Medical Investigation of Neurodevelopmental Disorders Institute, University of California at Davis Medical Center, Sacramento, CA 95817, USA

Hôpital de la Conception, Assistance Publique-Hôpitaux de Marseille, Boulevard Baille, Marseille F-13385, France

Current address: Genomics Institute of the Novartis Research Foundation, John Jay Hopkins Drive, San Diego, CA 92121, USA

 Genome Biology 1465-6906 2008 9 1 R17 http://genomebiology.com/2008/9/1/R17 18218067 10.1186/gb-2008-9-1-r17 28 8 2007 19 12 2007 24 1 2008 24 01 2008 2008 Robbins et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Profiling dendritic cell subsets

Genome-wide expression profiling of mouse and human leukocytes reveal conserved transcriptional programs of plasmacytoid or conventional dendritic cell subsets.

Abstract

Background

Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8α in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes.

Results

We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively.

Conclusion

Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.

 Immunology Evolution

Background

Dendritic cells (DCs) were initially identified by their unique ability to present antigen for the priming of naïve CD4 and CD8 T lymphocytes 1. DCs have more recently been shown to be key sentinel immune cells able to sense, and respond to, danger very early in the course of an infection due to their expression of a broad array of pattern recognition receptors 2. Indeed, DCs have been shown to play a major role in the early production of effector antimicrobial molecules such as interferon (IFN)-α and IFN-β 3 or inducible nitric oxide synthase 4 and it has been demonstrated that DCs can also activate other innate effector cells such as natural killer (NK) cells 5. In light of these properties, it has been clearly established that DCs are critical for defense against infections, as they are specially suited for the early detection of pathogens, the rapid development of effector functions, and the triggering of downstream responses in other innate and adaptive immune cells.

DCs can be divided into several subsets that differ in their tissue distribution, their phenotype, their functions and their ontogeny 6. Lymph node-resident DCs (LN-DCs) encompass conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in both humans and mice. LN-cDCs can be subdivided into two populations in both mouse (CD8α and CD11b cDCs) 6 and in human (BDCA1 and BDCA3 cDCs) 7. In mouse, CD8α cDCs express many scavenger receptors and may be especially efficient for cross-presenting antigen to CD8 T cells 8 whereas CD11b cDCs have been suggested 910, and recently shown 11, to be specialized in the activation of CD4 T cells. As human cDC functions are generally studied with cells derived in vitro from monocytes or from CD34+ hematopoietic progenitors, which may differ considerably from the naturally occurring DCs present in vivo, much less is known of the eventual functional specialization of human cDC subsets. Due to differences in the markers used for identifying DC subsets between human and mouse and to differences in the expression of pattern recognition receptors between DC subsets, it has been extremely difficult to address whether there are functional equivalences between mouse and human cDC subsets 6.

pDCs, a cell type discovered recently in both human and mouse, appear broadly different from the other DC subsets to the point that their place within the DC family is debated 3. Some common characteristics between human and mouse pDCs that distinguish them from cDCs 3 include: their ability to produce very large amounts of IFN-α/β upon activation, their limited ability to prime naïve CD4 and CD8 T cells under steady state conditions, and their expression of several genes generally associated with the lymphocyte lineage and not found in cDCs 12. Several differences have also been reported between human and mouse pDCs, which include the unique ability of mouse pDCs to produce high levels of IL-12 upon triggering of various toll-like receptors (TLRs) or stimulation with viruses 1314. Adding to the complexity of accurately classifying pDCs within leukocyte subsets are recent reports describing cell types bearing mixed phenotypic and functional characteristics of NK cells and pDCs in the mouse 1516. Collectively, these findings raise the question of how closely related human and mouse pDCs are to one another or to cDCs as compared to other leukocyte populations.

Global transcriptomic analysis has recently been shown to be a powerful approach to yield new insights into the biology of specific cellular subsets or tissues through their specific gene expression programs 1718192021. Likewise, genome-wide comparative gene expression profiling between mouse and man has recently been demonstrated as a powerful approach to uncover conserved molecular pathways involved in the development of various cancers 222324252627. However, to the best of our knowledge, this approach has not yet been applied to study normal leukocyte subsets. Moreover, DC subsets have not yet been scrutinized through the prism of gene expression patterns within the context of other leukocyte populations. In this report, we assembled compendia comprising various DC and other leukocyte subtypes, both from mouse and man. Using intra- and inter-species comparisons, we define the common and specific core genetic programs of DC subsets.

Results

Generation/assembly and validation of the datasets for the gene expression profiling of LN-DC subsets

We used pan-genomic Affymetrix Mouse Genome 430 2.0 arrays to generate gene expression profiles of murine splenic CD8α (n = 2) and CD11b (n = 2) cDCs, pDCs (n = 2), B cells (n = 3), NK cells (n = 2), and CD8 T cells (n = 2). To generate a compendium of 18 mouse leukocyte profiles, these data were complemented with published data retrieved from public databases, for conventional CD4 T cells (n = 2) 28 and splenic macrophages (n = 3) 29. We used Affymetrix Human Genome U133 Plus 2.0 arrays to generate gene expression profiles of blood monocytes, neutrophils, B cells, NK cells, and CD4 or CD8 T cells 30. These data were complemented with published data on human blood DC subsets (pDCs, BDCA1 cDCs, BDCA3 cDCs, and lin-CD16+HLA-DR+ cells) retrieved from public databases 31. All of the human samples were done in independent triplicates. Information regarding the original sources and the public accessibility of the datasets analyzed in the paper are given in Table 1.

<p>Table 1</p>

Information on the sources and public access for the datasets analyzed in the paper

Figures‡

Dataset

Population*

Laboratory†

Public repository

Accession number

1a,c; 2a

1b,d; 2b

1e

3

4a

4b

5a

5b

Affymetrix Mouse Genome 430 2.0 data

Spleen CD8 DCs (2)

MD/SCPK

GEO [95]

GSE9810

X

X

X

X

X

Spleen CD11b DCs (2)

MD/SCPK

GEO

GSE9810

X

X

X

X

X

Spleen pDCs (2)

MD/SCPK

GEO

GSE9810

X

X

X

X

X

Spleen NK cells (2)

MD/SCPK

GEO

GSE9810

X

X

X

Spleen CD8 T cells (2)

MD/SCPK

GEO

GSE9810

X

X

Spleen B cells (3)

MD/SCPK

GEO

GSE9810

X

X

X

Spleen CD4 T cells (2)

AYR

GEO

GSM44979; GSM44982

X

X

X

Spleen monocytes (3)

SB

NCI caArray [96]

NA

X

X

X

Spleen monocytes (2)

BP

GEO

GSM224733; GSM224735

X

Peritoneal MΦ (1)

SA

GEO

GSM218300

X

BM-MΦ (2)

RM

GEO

GSM177078; GSM177081

X

BM-MΦ (1)

CK

GEO

GSM232005

X

BM-DCs (2)

RM

GEO

GSM40053; GSM40056

X

BM-DCs (2)

MH

GEO

GSM101418; GSM101419

X

Affymetrix Mouse U74Av2 data

Spleen CD4 T cells (3)

CB/DM

GEO

GSM66901; GSM66902; GSM66903

X

Spleen B2 cells (2)

CB/DM

GEO

GSM66913; GSM66914

X

Spleen B1 cells (2)

CB/DM

GEO

GSM66915; GSM66916

X

Spleen NK cells (2)

FT

EBI ArrayExpress [97]

E-MEXP-354

X

Spleen CD4 DCs (2)

CRES

GEO

GSM4697; GSM4707

X

Spleen CD8 DCs (2)

CRES

GEO

GSM4708; GSM4709

X

Spleen DN DCs (2)

CRES

GEO

GSM4710; GSM4711

X

Spleen IKDCs (2)

FH

GEO

GSM85329; GSM85330

X

Spleen cDCs (2)

FH

GEO

GSM85331; GSM85332

X

Spleen pDCs (2)

FH

GEO

GSM85333; GSM85334

X

Affymetrix Human Genome U133 Plus 2.0 data

Blood monocytes (3)

FRS

Authors' webpage [86]

NA

X

X

X

X

Blood CD4 T cells (3)

FRS

Authors' webpage

NA

X

X

X

Blood CD8 T cells (3)

FRS

Authors' webpage

NA

X

X

X

Blood B cells (3)

FRS

Authors' webpage

NA

X

X

X

Blood NK cells (3)

FRS

Authors' webpage

NA

X

X

X

Blood neutrophils (3)

FRS

Authors' webpage

NA

X

X

X

Blood pDCs (3)

CAKB

EBI ArrayExpress

E-TABM-34

X

X

X

X

X

Blood BDCA1 DCs (3)

CAKB

EBI ArrayExpress

E-TABM-34

X

X

X

X

X

Blood BDCA3 DCs (3)

CAKB

EBI ArrayExpress

E-TABM-34

X

X

X

X

X

Blood CD16 DCs (3)

CAKB

EBI ArrayExpress

E-TABM-34

X

X

PBMC-derived MΦ (2)

SYH

GEO

GSM109788; GSM109789

X

Monocyte-derived MΦ

LZH

GEO

GSM213500

X

Monocyte-derived DCs (3)

MVD

GEO

GSM181931; GSM181933; GSM181971

X

*The number of replicates is shown in parentheses. †MD/SCPK, M Dalod, S Chan, P Kastner; AYR, AY Rudensky; SB, S Bondada; BP, B Pulendran; SA, S Akira; RM, R Medzhitov; CK, C Kim; MH, M Hikida; CB/DM, C Benoist, D Mathis; FT, F Takei; CRES, C Reis e Sousa; FH, F Housseau; FRS, FR Sharp; CAKB, CAK Borrebaeck; SYH, S Yla-Herttuala; LZH, L Ziegler-Heitbrock; MVD, MV Dhodapkar. ‡Shown in the indicated figure in this study. BM-DC, mouse bone-marrow derived GM-CSF DCs; BM-MΦ, mouse bone marrow-derived M-CSF macrophages; monocyte-derived MΦ, monocyte-derived M-CSF macrophages; NA, not applicable; PBMC-derived MΦ, human peripheral blood mononuclear cell-derived M-CSF macrophages; peritoneal MΦ, peritoneal mouse macrophages.

To verify the quality of the datasets mentioned above, we analyzed signal intensities for control genes whose expression profiles are well documented across the cell populations under consideration. Expression of signature markers were confirmed to be detected only in each corresponding population (see Table 2 for mouse data and Table 3 for human data). For example, Cd3 genes were detected primarily in T cells and often to a lower extent in NK cells; the mouse Klrb1c (nk1.1) gene or the human KIR genes in NK cells; Cd19 in B cells; the mouse Siglech and Bst2 genes or the human LILRA4 (ILT7) and IL3RA (CD123) genes in pDCs; and Cd14 in myeloid cells. As expected, many markers were expressed in more than a single cell population. For example, in the mouse, Itgax (Cd11c) was found expressed to high levels in NK cells and all DC subsets; Itgam (Cd11b) in myeloid cells, NK cells, and CD11b cDCs; Ly6c at the highest level in pDCs but also strongly in many other leukocyte populations; and Cd8a in pDCs and CD8α cDCs. However, the analysis of combinations of these markers confirmed the lack of detectable cross-contaminations between DC subsets: only pDCs expressed high levels of Klra17 (Ly49q) and Ly6c together, while Cd8a, ly75 (Dec205, Cd205), and Tlr3 were expressed together at high levels only in CD8α cDCs, and Itgam (Cd11b) with Tlr1 and high levels of Itgax (Cd11c) only in CD11b cDCs. Thus, each cell sample studied harbors the expected pattern of expression of control genes and our data will truly reflect the gene expression profile of each population analyzed, without any detectable cross-contamination.

<p>Table 2</p>

Expression of control genes in mouse cells

Dendritic cells

Lymphocytes

Probe set ID

Gene

Myeloid cells

pDC

CD8α DC

CD11b DC

NK

CD8 T

CD4 T

B

1419178_at

Cd3g

40 ± 10

<20

<20

<20

97 ± 31

2,074 ± 287

1,974 ± 478

22 ± 3

1422828_at

Cd3d

111 ± 14

<20

<20

<20

214 ± 16

2,815 ± 11

4,520 ± 1,414

21 ± 2

1422105_at

Cd3e

115 ± 30

27 ± 10

22 ± 2

23 ± 5

26 ± 9

387 ± 58

522 ± 210

26 ± 10

1426396_at

Cd3z

<20

<20

<20

<20

1,147 ± 81

1,545 ± 10

2,117 ± 482

25 ± 9

1426113_x_at

Tcra

83 ± 8

<20

23 ± 4

<20

116 ± 39

2,517 ± 42

5,601 ± 1,818

34 ± 13

1419696_at

Cd4

24 ± 2

1,233 ± 144

<20

369 ± 49

<20

<20

1,052 ± 73

<20

1450570_a_at

Cd19

190 ± 44

<20

<20

<20

<20

<20

23 ± 5

2,259 ± 292

1449570_at

Klrb1c (NK1.1)

<20

<20

<20

<20

2,328 ± 112

<20

25 ± 7

<20

1425436_x_at

Klra3 (Ly49C)

130 ± 11

24 ± 3

156 ± 0

242 ± 31

9,186 ± 479

170 ± 61

70 ± 42

<20

1450648_s_at

H2-Ab1

6,887 ± 84

7,339 ± 5

9,101 ± 100

9,056 ± 277

81 ± 6

83 ± 56

978 ± 11

7,028 ± 239

1419128_at

Itgax (CD11c)

454 ± 5

1,928 ± 169

2,827 ± 454

4,701 ± 56

3,403 ± 45

108 ± 44

22 ± 2

<20

1457786_at

Siglech

31 ± 4

3,454 ± 536

24 ± 5

<20

<20

<20

33 ± 13

<20

1425888_at

Klra17 (Ly49Q)

98 ± 4

3,413 ± 116

30 ± 14

163 ± 2

28 ± 11

24 ± 6

38 ± 10

<20

1424921_at

Bst2 (120G8)

2,364 ± 149

5,571 ± 718

237 ± 30

196 ± 44

61 ± 24

162 ± 12

90 ± 3

88 ± 32

1421571_a_at

Ly6c

4,420 ± 261

8,255 ± 151

98 ± 5

30 ± 8

2,082 ± 365

4,530 ± 229

1,789 ± 1,242

302 ± 303

1422010_at

Tlr7

439 ± 13

846 ± 40

<20

322 ± 45

<20

<20

22 ± 2

118 ± 83

1440811_x_at

Cd8a

<20

337 ± 134

825 ± 44

<20

<20

1,235 ± 227

22 ± 2

<20

1449328_at

Ly75 (Dec205)

249 ± 27

<20

159 ± 4

22 ± 3

24 ± 6

170 ± 29

79 ± 1

21 ± 1

1422782_s_at

Tlr3

27 ± 2

25 ± 3

3,376 ± 159

287 ± 14

<20

<20

<20

52 ± 45

1422046_at

Itgam (CD11b)

956 ± 57

<20

<20

162 ± 1

188 ± 38

<20

<20

21 ± 1

1449049_at

Tlr1

1,218 ± 54

31 ± 15

101 ± 4

1,601 ± 92

<20

889 ± 109

498 ± 103

1,141 ± 484

1417268_at

Cd14

7,649 ± 169

187 ± 52

107 ± 0

115 ± 34

<20

<20

31 ± 8

27 ± 12

1449498_at

Marco

174 ± 19

<20

<20

<20

<20

<20

<20

<20

1460282_at

Trem1

415 ± 19

<20

<20

<20

<20

<20

<20

<20

 <p>Table 3</p>

Expression of control genes in human cells

Lymphocytes

Dendritic cells

Myeloid cells

Probe set ID

Genes

NK

CD8 T

CD4 T

B

pDC

BDCA1

BDCA3

Mono

Neu

206804_at

CD3G

858 ± 71

1,760 ± 241

1,975 ± 132

53 ± 6

<50

<50

<50

<50

52 ± 4

213539_at

CD3D

5,413 ± 238

7,134 ± 635

6,291 ± 285

276 ± 24

<50

<50

51 ± 2

112 ± 9

276 ± 4

205456_at

CD3E

247 ± 21

569 ± 67

679 ± 91

<50

<50

<50

<50

<50

<50

210031_at

CD3Z

8,688 ± 181

5,223 ± 218

4,749 ± 123

2,996 ± 217

56 ± 10

60 ± 17

54 ± 7

914 ± 96

132 ± 15

209671_x_at

TCR@

147 ± 16

3,127 ± 260

3,462 ± 170

71 ± 7

<50

<50

<50

<50

111 ± 16

205758_at

CD8A

911 ± 26

5,259 ± 217

67 ± 10

79 ± 16

<50

<50

<50

<50

99 ± 7

207979_s_at

CD8B

77 ± 9

3,596 ± 299

<50

<50

<50

<50

<50

<50

53 ± 5

203547_at

CD4

<50

<50

391 ± 20

83 ± 20

1,301 ± 119

1,004 ± 74

278 ± 61

205 ± 34

<50

206398_s_at

CD19

<50

51 ± 1

<50

1,726 ± 115

<50

<50

<50

57 ± 12

<50

212843_at

NCAM1 (CD56)

2,074 ± 96

144 ± 14

65 ± 2

135 ± 9

<50

<50

82 ± 17

52 ± 3

<50

207314_x_at

KIR3DL2

3,131 ± 172

454 ± 14

227 ± 18

265 ± 16

<50

<50

<50

59 ± 8

<50

208203_x_at

KIR2DS5

3,472 ± 140

444 ± 7

236 ± 10

284 ± 14

<50

<50

<50

<50

<50

239975_at

HLA-DPB2

<50

<50

<50

63 ± 22

777 ± 701

1,565 ± 519

2,056 ± 577

<50

<50

210184_at

ITGAX (CD11c)

1,017 ± 50

112 ± 37

166 ± 17

752 ± 45

74 ± 21

2,151 ± 430

729 ± 98

1,284 ± 115

2,133 ± 196

210313_at

LILRA4 (ILT7)

226 ± 10

117 ± 13

346 ± 42

1,109 ± 76

7,916 ± 612

230 ± 16

1,659 ± 1,183

524 ± 41

<50

206148_at

IL3RA (CD123)

84 ± 3

59 ± 8

91 ± 2

324 ± 9

4,728 ± 365

61 ± 10

116 ± 110

120 ± 3

74 ± 12

1552552_s_at

CLEC4C (BDCA2)

93 ± 6

61 ± 5

99 ± 4

408 ± 9

6,789 ± 737

76 ± 39

859 ± 434

217 ± 8

175 ± 25

205987_at

CD1C (BDCA1)

76 ± 8

61 ± 12

159 ± 8

1,715 ± 85

64 ± 23

8,313 ± 272

722 ± 845