Combination Of Mass Cytometry and Imaging

Accepted Manuscript Combination Of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice Bruna Araujo David, Rafael Machado Rezende, Maísa Mota Antunes, Mônica Morais Santos, Maria Alice Freitas Lopes, Ariane Barros Diniz, Rafaela Vaz Sousa Pereira, Sarah Cozzer Marchesi, Débora Moreira Alvarenga, Brenda Naemi Nakagaki, Alan Moreira Araújo, Daniela Silva dos Reis, Renata Monti Rocha, Pedro Elias Marques, Woo-Yong Lee, Justin Deniset, Pei Xiong Liew, Stephen Rubino, Laura Cox, Vanessa Pinho, Thiago Mattar Cunha, Gabriel Rocha Fernandes, André Gustavo Oliveira, Mauro Martins Teixeira, Paul Kubes, Gustavo Batista Menezes PII: DOI: Reference:

S0016-5085(16)34966-6 10.1053/j.gastro.2016.08.024 YGAST 60647

To appear in: Gastroenterology Accepted Date: 21 August 2016 Please cite this article as: David BA, Rezende RM, Antunes MM, Santos MM, Freitas Lopes MA, Diniz AB, Sousa Pereira RV, Marchesi SC, Alvarenga DM, Nakagaki BN, Araújo AM, dos Reis DS, Monti Rocha R, Marques PE, Lee W-Y, Deniset J, Liew PX, Rubino S, Cox L, Pinho V, Cunha TM, Fernandes GR, Oliveira AG, Martins Teixeira M, Kubes P, Menezes GB, Combination Of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice, Gastroenterology (2016), doi: 10.1053/j.gastro.2016.08.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Combination Of Mass Cytometry and Imaging Analysis Reveals Origin, Location, and Functional Repopulation of Liver Myeloid Cells in Mice

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Short title: Functional repopulation of liver phagocytes

Bruna Araujo David1, Rafael Machado Rezende2, Maísa Mota Antunes1, Mônica Morais Santos1, Maria Alice Freitas Lopes1, Ariane Barros Diniz1,

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Rafaela Vaz Sousa Pereira1, Sarah Cozzer Marchesi1, Débora Moreira Alvarenga1, Brenda Naemi Nakagaki1, Alan Moreira Araújo1, Daniela Silva

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dos Reis3, Renata Monti Rocha3, Pedro Elias Marques3, Woo-Yong Lee4, Justin Deniset4, Pei Xiong Liew4, Stephen Rubino2, Laura Cox2, Vanessa Pinho5, Thiago Mattar Cunha6, Gabriel Rocha Fernandes7, André Gustavo

Menezes1,*

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Oliveira1,8, Mauro Martins Teixeira3, Paul Kubes4,* and Gustavo Batista

1- Center for Gastrointestinal Biology, Departamento de Morfologia, Instituto

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de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 31270-901, Brazil. 2- Ann Romney Center for

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Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA. 3- Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 31270-901, Brazil. 4- University of Calgary, Alberta, T2N 4N1, Canada. 5-Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 31270-901, Brazil. 6- Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, 14049-900, Brazil. 7- Centro de

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ACCEPTED MANUSCRIPT Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Minas Gerais, 30190002, Brazil. 8- Departamento de Fisiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 31270-

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901, Brazil. Authors Positions:

B.A.D., M.M.A, A.B.D., D.M.A. and A.M.A are Ph.D. students in Universidade

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Federal de Minas Gerais, Brazil.

S.R. and L.C. are Post-doctoral researchers in Brigham and Women’s

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Hospital, Harvard Medical School, Harvard University, Boston, USA. R.M.R. is Instructor in Neurology at Harvard Medical School and Assistant Scientist at Brigham and Women’s Hospital, Harvard University, Boston, USA.

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W.Y.L., J.D. and P.X.L. are Post-doctoral researchers in University of Calgary, Canada.

M.M.S. and P.E.M. are Post-doctoral researchers at Federal University of

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Minas Gerais, Brazil.

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R.V.P.S., S.C.M., B.N.N. are Master Students at Universidade Federal de Minas Gerais, Brazil.

M.A.F.L. is a Bachelor in Biology undergrad at Universidade Federal de Minas Gerais, Brazil. D.S.R. is a Flow Cytometry technician at Universidade Federal de Minas Gerais, Brazil. G.R.F. is a Bioinformatician at Fundação Oswaldo Cruz, Brazil.

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ACCEPTED MANUSCRIPT P.K. is Professor at University of Calgary, Canada. V.P., A.G.O., M.M.T. and G.B.M are Professors in Universidade Federal de Minas Gerais and T.M.C. at Universidade de São Paulo, Brazil.

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* Authors in bold jointly supervised this project. Grant Support: This work was supported by FAPESP, FAPEMIG, CAPES (Biocomputacional) and CNPq (Brazil), and Canadian Institutes of Health

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Research and the Heart and Stroke Foundation of Canada.

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List of abbreviations:

BM: Bone marrow; CLL: clodronate-loaded liposomes; CyTOF: Time-of-flight mass cytometry; DCs: Dendritic cells; KCs: Kupffer cells; LNPC: Liver nonparenchymal cells; NK: Natural killer cell; NKT: Natural Killer T Cell

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Correspondence and requests for materials should be addressed to Gustavo B Menezes ([email protected]) or Paul Kubes ([email protected]).

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Authors have nothing to disclosure.

GEO accession number: GSE81645

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Author Contributions: B.A.D., R.M.R., M.M.A., M.M.S., M.A.F.L., A.B.D., R.V.S.P., S.C.M., D.M.A., B.N.M., A.M.A., P.E.M. and A.G.O. performed in vivo experiments and digital imaging analysis. D.S.R., S.R., L.C., G.R.F. performed liver cells isolation and characterization. R.M.P. performed mouse breeding and genetic analysis. W.Y.L., J.D. and P.X.L. performed part of intravital experiments, imaging quantification and helped to write the paper. V.P., T.M.C. and M.M.T. performed multiplexed cytokine analysis and revised

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ACCEPTED MANUSCRIPT the paper. B.A.D., R.M.R. G.B.M. and P.K. designed the study and wrote the paper. Acknowledgements: We thank Dr. Julio Scharfstein and Dr. Clarissa

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Nascimento for providing CD11c-EYFP mice (UFRJ, Brazil) and Dr Howard Weiner, Dr Shirong Liu (Harvard Medical School, USA), Giuliana Bertozi (FMRP, Universidade de São Paulo, Brazil) and Dr. Flaviano Martins (UFMG, Brazil) for assistance, reagents and mice strains. We thank Rodrigo Nogueira,

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Paulo Lutero de Mello e Silva II and Dr. Denise Cara for helping in the

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generation of scientific divulgation material. Also, we would like to thank the Centro de Aquisição e Processamento de Imagens (CAPI- ICB/UFMG) and the Flow Cytometry facility of Centro de Pesquisas René Rachou (FIOCRUZ-

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MG).

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ACCEPTED MANUSCRIPT Abstract: Background & Aims: Resident macrophages are derived from yolk sac precursors and seed the liver during embryogenesis. Native cells may be

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replaced by bone marrow precursors during extensive injuries, irradiation and infections. We investigated the liver populations of myeloid immune cells and their location as well as the dynamics of phagocyte repopulation after full depletion. The effects on liver function due to the substitution of original

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phagocytes by bone marrow-derived surrogates were also examined.

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Methods: We collected and analyzed liver tissues from C57BL/6 (control), LysM-EGFP, B6 ACTb-EGFP, CCR2-/-, CD11c-EYFP, CD11c-EYFP-DTR, Germ-free mice, CX3CR1gfp/gfp, CX3CR1gpf/wt and CX3CR1-DTR-EYFP. Liver non-parenchymal cells were immunophenotyped using mass cytometry and

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gene expression analyses. Kupffer and dendritic cells were depleted from mice by administration of clodronate, and their location and phenotype were examined using intravital microscopy and time-of-flight mass cytometry. Mice

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were given acetaminophen gavage or intravenous injections of fluorescently labeled Escherichia coli, blood samples were collected and analyzed, and

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liver function was evaluated. We assessed cytokine profiles of liver tissues using a multiplexed array. Results: Using mass cytometry and gene expression analyses, we identified 2 populations of hepatic macrophages and 2 populations of monocytes. We also identified 4 populations of dendritic cells and 1 population of basophils. After selective depletion of liver phagocytes, intravascular myeloid precursors began to differentiate into macrophages and dendritic cells; dendritic cells

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ACCEPTED MANUSCRIPT migrated out of sinusoids, after a delay, via the chemokine CX3CL1. The cell distribution returned to normal in 2 weeks, but the repopulated livers were unable to fully respond to drug-induced injury or clear bacteria for at least 1 month. This defect was associated with increased levels of inflammatory

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cytokines, and dexamethasone accelerated the repopulation of liver phagocytes.

Conclusions: In studies of hepatic phagocyte depletion in mice, we found

which

each

localize

to

distinct

tissue

compartments.

During

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cells,

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that myeloid precursors can differentiate into liver macrophages and dendritic

replenishment, macrophages acquire the ability to respond appropriately to hepatic injury and to remove bacteria from the blood stream.

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KEY WORDS: CyTOF; DC; liver development; mouse model

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ACCEPTED MANUSCRIPT

Introduction

The liver is a key organ for immune maturation during embryogenesis

1, 2

and

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also plays an essential role in immune surveillance throughout life. The hepatic environment physiologically harbors a vast population of immune cells, including NK

3

and NKT cells 4, dendritic cells (DCs)

5

and macrophages

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(Kupffer cells; KCs) 6. In order to accomplish critical innate immune functions,

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phagocytic cells are strategically positioned within hepatic compartments. KCs are located in the sinusoidal lumen where they are constantly surveying blood content, ingesting aging erythrocytes

7, 8

and catching pathogens out of the

mainstream of blood 9, 10. Recent studies have shown that macrophages seed the liver during embryogenesis from yolk sac progenitors, and this resident pool may be maintained in adulthood via both self-renewal of precursors and to a lesser extent recruitment of bone marrow (BM)-derived cells. Indeed there is a

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ACCEPTED MANUSCRIPT growing body of evidence that the KC population is mainly composed of cells related to embryonic precursors rather than hematopoietic cells

11, 12

. DCs

develop from bone marrow-derived hematopoietic stem cells, but circulating monocyte precursors can also contribute to the resident DC pool

. A small

14-16

; however, in vivo

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population of liver DCs have been previously described

13

studies aiming to define the precise location and actual density of DCs within the liver, as well as their relationship with other hepatic cells, are needed.

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destroy whole pathogens remains debatable 17.

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Additionally, despite being described as phagocytes, their ability to engulf and

The resident DC and KC liver populations in adulthood therefore are some combination of embryonic-derived precursors and monocyte-derived cells, and it is likely that multiple factors define the extent of the contribution of these two sources 2. However, during “emergency repopulation” – a term we

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coined for the sudden complete loss of immune cells due to toxins, pathogens (i.e., Listeria), or trauma – self-replication may not be an option. Here we used

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a combination of high-throughput analysis with live imaging approaches to precisely determine phagocytic populations within the liver and the functional

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consequences of their replenishment by bone marrow cells.

Results

High dimensional identification of liver phagocytes To better characterize the resident phagocytic populations, we purified liver non-parenchymal cells (CD45+ LNPCs) and performed time-of-flight mass

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ACCEPTED MANUSCRIPT cytometry (CyTOF) analysis. CyTOF allows for simultaneous staining with multiple combinations of cell markers, and can reveal new immune cell populations that would not otherwise be uncovered by conventional flow cytometry. In these experiments, we gated on CD45+ cells and excluded red

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blood cells, Ito cells and B and T cells during analysis. Using an unbiased viSNE analysis to map high-dimensional cytometry data, we identified twelve clusters of LNPCs (Figure 1A). The majority of cells mapped to either F4/80

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(clusters 1-4) or CD11c (clusters 5-8) (Figure 1A and B). Cluster 1 was defined as mature granulocytes due to intermediate expression level of Ly6G

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(Figure 1A and B) and high expression of CD24 (Supplementary Figure 1A). Based on the expression of F4/80, MHCII and CD11b (Figure 1A and B), we defined two different populations of Kupffer cells (clusters 2 and 3). KC cluster 2 expressed CD206, CD317 and CD1d whereas cluster 3 was negative for

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CD206, CD317 and CD1d, but expressed CD11c (Figure 1A and Supplementary Figure 1A). Cluster 4 and 5 were characterized as infiltrating monocytes, since they were CD11b+Ly6C+MHC-IintMHC-IIlo, but cluster 5 was

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F4/80- and CD11clo (Figure 1A and B). Clusters 6-8 and 11 were categorized

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as DCs, since they were CD11c+CD11bint/hiF4/80-Ly6G-MHC-I+MHC-II+/lo. Cluster 8 was defined as plasmacytoid dendritic cells (pDCs) due to their high expression of the pDC marker CD317 (Figure 1A and B). Cluster 9 was also defined as granulocytes according to their high expression of Ly6G, CD11b and Gr1 as well as intermediate expression of CD24 and Ly6C (Figure1A and 1B and Supplementary Figure 1A). Finally, cells that expressed the high affinity IgE receptor (FcεRI), together with CD44 and CD11b, were clustered as basophils (cluster 10) (Figure 1A and B and Supplementary

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ACCEPTED MANUSCRIPT Figure 1A). Cluster 11 may represent a population of conventional DCs due to the expression of CD11c, CD11b, CD8a and CD103 (Figure 1A and B and Supplementary Figure 1A). CD11c+ NK cells (NK1.1 and NKp46) comprised an insignificant population in this setup (5). Supplementary Figure 4 Liver dendritic cells are located in the subcapsular compartment and are CD11c+ (A) Liver intravital microscopy of CX3CR1gfp/wt mouse showing the distribution of CD11c+ dendritic (anti-CD11) cells and

ACCEPTED MANUSCRIPT (B,C) confirmation of sub-mesothelial location of CX3CR1+ cells by intravital microscopy (B) immunohistochemistry (C). Illustrative images from different experiments (N>5).

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Supplementary Figure 5 Characterization of different cell surface markers and CCR2 role in phagocytes (A) Liver ex vivo transversal fragment showing that subcapsular neither CX3CR1+ nor CD11c+ cells are positive for desmin

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(anti-desmin antibody), excluding this population as hepatic stellate cells. (B) Real-time PCR from isolated macrophages and dendritic cells showing that

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hepatic stellate cells (HSCs) did not contaminate samples. The following HSCs genes were measured: lecithin retinol acyltransferase (Lrat), desmin (Des) and alpha-actin-2 (Acta2). Data were normalized by a constitutive gene (Gapdh) and Itgax (CD11c) was used as positive control. (C) Liver intravital microscopy showing that neither CX3CR1+ cells nor F4/80+ have vitamin A

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granules (auto-fluorescence in 405nm laser). (D) Flow cytometry investigation of the absence of vitamin A in CX3CR1+ population (auto-fluorescence in 405nm laser). (E) CCR2-/- mouse have normal KC density (anti-F4/80) and (F)

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location. Scale bars in A, 10 µm; B, 65µm and E, 120µm.

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Supplementary Figure 6 Additional dot plots from CyTOF analysis from mice 7 and 17 days after phagocyte depletion. Expression of cell surface markers in liver non-parenchymal cells clodronate depleted mice after 7 days (A) and after 17 days (B). CyTOF was performed and events were clustered as described in Methods and Results. Supplementary Figure 7 (A) Replenishment of liver phagocytes is driven by a bone marrow-derived precursor. Different focal planes imaged by intravital

ACCEPTED MANUSCRIPT confocal microscopy from GFP-expressing bone marrow chimeras showing that all Kupffer cells (F4/80+GFP+ cells in intravascular focus) and dendritic cells (GFP+ cells in capsule focus) were derived from the bone marrow. (B) Acetaminophen (APAP) treatment caused a significant depletion of KCs (red;

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F4/80+ cells) and normal cell density and location were restored after 17-30 days. In blue, DAPI stains for necrosis. Scale bars in KC focus, 35 µm, in DC focus, 20 µm and in APAP-treated group 120 µm. *P < 0.05 (unpaired t-test)

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Legends for Tables

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in comparison to vehicle.

Table 1 Antibody panel for CyTOF and for genes for Real Time PCR. For PCR, cells were isolated and RNA was extracted with a miRNeasy kit

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(Qiagen), then was reverse-transcribed with a high capacity cDNA reverse transcription kit and analyzed by quantitative RT-PCR with a Vii 7 Real-time PCR system with Taqman primers. The following HSCs genes were

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measured: lecithin retinol acyltransferase (Lrat), desmin (Des) and alphaactin-2 (Acta2). Data were normalized by a constitutive gene (Gapdh) and

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Itgax (CD11c) was used as positive control. The comparative threshold cycle method and the internal control Gapdh was used for normalization of the target genes.

Table 2 Absolute cell numbers (events) from time-of-flight flow cytometry (CyTOF) experiments. Liver samples from different groups were collected and processed for CyTOF. Events were normalized in all samples.

ACCEPTED MANUSCRIPT Table 3 Quantification of liver cytokine expression by multiplexed Luminex array. Liver samples from different groups were collected and processed for multiplexed cytokine array. Data are displayed as individual samples from

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different experiments. GEO accession number: GSE81645. Gene expression assessed by Nanostring experiments. Intravascular CX3CR1- F4/80+ and extravascular

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CX3CR1+ F4/80- cells were isolated by sorting (FACS) and immune systemrelated genes expression was quantified using Nanostring. Statically relevant

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results consist in p-value < 0.05 and a fold change of at least 50% higher or lower. Pathways and functional classification were done by cross association using KEGG Pathways and KEGG Brite databases.

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Legends for Supplementary Movies

Supplementary Movie 1 Identification of main hepatic vascular arrangements. Temporal distribution of FITC-albumin (green) within the liver (5mg/Kg). Portal

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spaces were identified as the first vascular region evidenced by fluorescence,

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and central vein were defined as the secondary draining vessels. Total experimenta time: 1 minute. Supplementary movie 2 Three-dimensional rendering of liver intravital imaging. Spatial distribution of Kupffer cells. Vessels are in blue (anti-PECAM1 antibody) and KCs in red (anti-F4/80 antibody). Images were collect from a 3D section of 80-100µm of depth.

ACCEPTED MANUSCRIPT Supplementary movie 3 Three-dimensional rendering of liver intravital imaging. Liver dendritic cell (DCs) morphology and distribution in CD11cEYFP mice. Vessels are in blue (anti-PECAM-1 antibody) and DCs in yellow

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(EYFP). Images were collect from a 3D section of 80-100µm of depth.

Supplementary movie 4 Three-dimensional rendering of liver intravital

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imaging. Liver dendritic cell morphology and distribution in CX3CR1-EGFP mice. Vessels are in blue (anti-PECAM-1 antibody) and DCs in green (EGFP).

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Images were collect from a 3D section of 80-100µm of depth.

Supplementary movie 5: KCs instantaneously trap circulating E. coli from the circulation. Merged video comparing E. coli arresting by a control KC (non-depleted mouse) versus an immature KC (7 days after CLL). Note that E.

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coli are arrested at the first passage through the liver, while immature KC are unable to proper catch and internalize bacteria. Kupffer cells (KCs) are in red and E. coli in green. Total video time: 10 minutes. 107 E.coli were injected in

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the beginning of the imaging procedure.

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Supplementary movie 6 Circulating E. coli capture by Kupffer cells. Merged video showing E. coli arresting by control group (non-depleted mouse) and after different timepoints of replenishment period (7, 17, 30 and 60 days). Kupffer cells (KCs) are in red and E. coli in green. Due to photobleaching issues, a still frame from KC channel was used as reference during the video. Total video time: 10 minutes each experimental group. 107 E.coli were injected in the beginning of the imaging procedure

ACCEPTED MANUSCRIPT Supplementary movie 7 E. coli are exclusively arrested by intravascular cells Three-dimensional rendering of liver confocal intravital microscopy showing GFP expressing E coli inside sinusoids. Sinusoids are in blue (anti-PECAM-1 antibody) and E. coligfp in green. Images were collect from a 3D section of 80-

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100µm of depth.

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N418 927 10F.9G2 H57-597 HK1.4

145Nd 146Nd 148Nd 149Sm 150Nd 151Eu

CD3 CD172a/SIRPa CD103 CD106/VCAM-1

145-2C11 P84 2.00E+07 429

152Sm 153Eu 154Sm 155Gd

CD19 Ly6G

6D5 1A8

156Gd 158Gd

CD11b CD54/ICAM-1

M1/70 YN1/1.7.4 M1/69 53-6.7 30-F11 GL-1

160Gd 161Dy

167Er 169Tm

CD80 CD206 H-2Ld/H-2Db (MHC I)

3C7 MAR*1 Mar-23 C068C2 28-14-8

I-A/I-E (MHC II) F4/80

M5/114.15.2 BM8

174Yb 175Lu

CD24 CD8a CD45 CD86/B7-2

170Er 172Yb 173Yb

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CD25/IL-2Ra FCeR1

162Dy 164Dy 165Ho 166Er

Genes for RT-PCR (Taqman Probes) Lrat - Mm00469972_m1 Acta2 - Mm01546133_m1 Des - Mm00802455_m1 Itgax - Mm00498701_m1 Gapdh (Mm99999915-g1)

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CD4 CD11c CD317/PDCA-1 CD274 (PDL-1) TCRb Ly6C

139La 141Pr 142Nd 143Nd 144Nd

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CD44 CD115 TER-119 CD1d

CLONE RB6-8C5 IM7 AFS98 TER-119 K253 RM4-5

MANUSCRIPT

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ANTIGEN GR1

ACCEPTED METALCONJUGATE

ACCEPTED MANUSCRIPT F4/80+

without CLL day 2 day 7 day 17

Cluster 4 without CLL day 2 day 7 day 17

mouse 4

AVERAGE

1300 1620 1300 800

1520 1370 1220 1900

1637.5 1457.5 1477.5 1395

mouse 1

mouse 2

mouse 3

mouse 4

AVERAGE

1090 90 670 600

370 180 870 570

990 50 1660 720

670 80 910 640

mouse 1

mouse 2

mouse 3

mouse 4

1710 1670 4960 1500

1460 1230 4160 2180

1790 1510 3290 2110

2230 1690 2530 2070

1797.5 1525 3735 1965

mouse 1

mouse 2

mouse 3

mouse 4

AVERAGE

1420 6420 1610 4280

1650 6380 2300 3680

1780 6700 1910 3730

1390 6670 1220 1660

1560 6542.5 1760 3337.5

mouse 1

mouse 2

mouse 3

mouse 4

AVERAGE

2640 1650 1590 2890

2470 1810 2610 3670

2250 1720 1340 2570

2270 1600 1330 2200

2407.5 1695 1717.5 2832.5

mouse 2





mouse 1

780 100 1027.5 680

AVERAGE

mouse 3

mouse 4

AVERAGE

10270 6400 6370 2780

9090 7350 4390 880

9720 2780 9010 7020

8300 1860 10040 8590

9345 4597.5 7452.5 7805

mouse 1

mouse 2

mouse 3

mouse 4

AVERAGE

4170 3680 3820 5370

6330 4800 3150 5680

4720 2600 4210 4350

4490 1910 4450 4960

4927.5 3247.5 3907.5 5090

mouse 1

mouse 2

mouse 3

mouse 4

AVERAGE

1410 3960 3340 1850

1950 3720 4250 1540

1810 3900 3010 1380

2190 4090 2560 2510

1840 3917.5 3290 1820

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Cluster 5

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CD11c+

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Cluster 3

mouse 3

2030 1730 1440 1890

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mouse 2

1700 1110 1950 990

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Cluster 2

mouse 1

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ACCEPTED MANUSCRIPT mouse 3 mouse 4

mouse 1

mouse 2


900 1120 2030 550

620 730 2220 1050

1200 1250 1460 1280

AVERAGE

1700 1360 1050 670

1105 1115 1690 887.5

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EP

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SC

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Cluster 11

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Luminex Assay G-CSF pg/ml

Eotaxin pg/ml

GM-CSF pg/ml

IFN-gama pg/ml

IL-1alfa pg/ml

IL-1beta pg/ml

IL-2 pg/ml

IL-4 pg/ml

IL-3 pg/ml

IL-5 pg/ml

IL-6 pg/ml

IL-7 pg/ml

IL-9 pg/ml

IL-10 pg/ml

IL-12 (p40) pg/ml

IL-12 (p70) pg/ml

LIF pg/ml

IL-13 pg/ml

LIX pg/ml

IL-15 pg/ml

IL-17 pg/ml

IP-10 pg/ml

KC pg/ml

MCP-1 pg/ml

MIP-1alfa pg/ml

MIP-1beta pg/ml

M-CSF pg/ml

MIP-2 pg/ml

MIG pg/ml

RANTES pg/ml

VEGF pg/ml

TNF-alfa pg/ml

Control 1 Control 2 Control 3 CLL day 1 - 1 CLL day 1 - 2 CLL day 1- 3 CLL day 1 - 4 CLL day 2 - 1 CLL day 2 - 2 CLL day 2 - 3 CLL day 2 - 4 CLL day 7 - 1 CLL day 7 - 2 CLL day 7 - 3 CLL day 7 - 4 CLL dia 17 - 1 CLL dia 17 - 2 CLL dia 17 - 3

41.6 65.42 52.17 514.37 613.37 527.47 713.27 62.21 34.45 52.17 52.17 71.44 70.55 68.8 55.56 97.83 57.69 82.04

288.46 343.98 208.02 311.06 188.11 197.82 161.77 583.29 318.98 213.32 339.68 292.11 202.85 178.89 146.29 311.06 241.9 284.85

81.01 82.04 74.19 81.01 63.8 60.67 30.77 79 41.6 40.57 54.86 83.08 119.63 118.13 60.67 122.67 118.13 122.67

119.63 216.02 90.72 241.9 554.67 370.93 251.2 213.32 202.85 453.59 213.32 95.4 84.13 82.04 132.29 129 102.87 303.34

192.9 248.06 235.89 395 274.31 235.89 230.03 205.42 335.44 260.85 232.94 295.8 192.9 260.85 208.02 295.8 254.38 327.11

127.39 185.76 157.75 176.65 132.29 139.11 70.55 157.75 79 140.87 110.93 157.75 238.88 257.59 132.29 303.34 241.9 390.07

32.35 31.95 31.95 54.86 36.69 32.76 43.2 31.95 40.57 40.06 33.6 26.79 23.33 21.64 29.63 32.76 29.63 34.45

6.38 8.9 6.29 12.94 8.39 7.47 8.64 9.44 11.08 7.69 10.01 7.05 4.09 5.95 5.07 9.44 6.29 8.52

10.01 19.57 14.43 17.69 9.58 14.05 4.59 25.8 10.76 14.43 15.2 23.93 30 27.82 44.86 22.19 12.76 40.57

27.47 27.13 30 32.35 30.77 25.8 14.43 63.8 30 27.82 44.86 34.89 32.35 43.2 32.35 68.8 47.18 54.86

254.38 318.98 318.98 514.37 410.19 390.07 370.93 318.98 361.72 380.38 318.98 303.34 218.75 267.5 274.31 335.44 281.29 343.98

74.19 113.76 90.72 102.87 76.08 68.8 41.6 84.13 52.17 76.08 68.8 113.76 95.4 115.2 86.27 150.01 129 183.44

1517 1656 1406 1878 1498 1389 1555 1517 1517 1720 1720 1831 1225 1389 1272 1720 1288 1720

142.65 165.89 142.65 213.32 122.67 108.18 113.76 121.14 178.89 161.77 144.46 135.66 110.93 190.49 125.8 165.89 142.65 176.65

165.89 208.02 213.32 331.25 254.38 241.9 188.11 208.02 157.75 224.32 224.32 284.85 260.85 274.31 200.32 251.2 218.75 299.55

122.67 110.93 102.87 202.85 150.01 119.63 50.88 116.66 53.5 100.32 139.11 88.47 97.83 86.27 81.01 197.82 165.89 218.75

1.72 4.09 3.13 13.67 21.1 15.6 14.81 4.48 10.01 2.45 3.39 18.61 15.8 12.59 4 6.12 5.63 5.34

184.03 219.45 193.52 282.19 179.46 175 144.92 179.46 222.22 216.7 193.52 156.28 150.49 184.03 143.11 214 175 225.04

343.98 425.95 274.31 489.14 425.95 284.85 335.44 235.89 352.74 235.89 339.68 1150 628.99 808.83 213.32 277.78 299.55 303.34

514.37 769.16 598.14 808.83 645.01 514.37 410.19 613.37 628.99 645.01 628.99 628.99 645.01 808.83 568.8 1014 695.55 1150

25.48 34.45 28.53 36.69 30.38 23.93 15.2 33.18 20.84 28.53 27.13 25.8 27.47 28.53 22.75 42.66 42.66 51.52

248.06 343.98 352.74 769.16 583.29 527.47 361.72 238.88 442.33 174.44 208.02 235.89 307.17 260.85 248.06 274.31 241.9 281.29

465.15 224.32 170.11 940.56 695.55 527.47 311.06 183.44 241.9 235.89 218.75 964.52 235.89 216.02 150.01 183.44 150.01 202.85

125.8 192.9 161.77 1809 1461 1014 621.13 202.85 79 170.11 146.29 590.67 299.55 311.06 323.02 251.2 221.52 288.46

96.61 178.89 90.72 216.02 195.34 197.82 64.6 257.59 153.83 159.75 267.5 99.07 178.89 218.75 190.49 299.55 254.38 135.66

125.8 59.16 41.6 142.65 99.07 161.77 60.67 41.6 25.16 26.46 23.93 29.26 50.88 44.86 197.82 62.21 50.88 74.19

113.76 183.44 174.44 380.38 311.06 224.32 135.66 213.32 60.67 142.65 161.77 105.49 132.29 170.11 105.49 489.14 335.44 390.07

216.02 267.5 238.88 327.11 311.06 295.8 192.9 307.17 216.02 284.85 251.2 205.42 230.03 270.88 227.16 420.63 352.74 436.8

1855 3292 3106 32860 22334 18695 29531 2405 2341 1902 2615 8832 9737 3925 3197 1926 1636 4945

50.88 48.38 48.38 54.86 52.17 41.6 30.77 35.78 62.21 32.35 35.33 34.89 39.56 39.07 53.5 38.58 41.6 52.17

20.06 34.02 24.53 79 47.78 36.23 43.75 43.75 62.21 35.78 42.66 70.55 67.09 27.82 24.53 25.16 27.13 33.18

5.79 24.53 11.56 17.69 13.3 13.67 2.73 19.57 3.29 19.57 9.17 15.2 27.82 27.82 11.56 40.57 30 41.6

Samples experiment 2 Control 1 Control 2 Control 3 Control 4 Control 5 CLL day 30 - 1 CLL day 30 - 2 CLL day 30 - 3 CLL day 30 - 4 CLL day 30 - 5 CLL day 30 - 6 CLL day 60 - 1 CLL day 60 - 2 CLL day 60 - 3 CLL day 60 - 4 CLL day 60 - 5 CLL day 60 - 6

47.2 35 31.6 83.2 37.7 41.4 96.1 150.1 72.6 62.1 60.7 33.6 29.6 28.9 43.4 30.9 49.5

987.5 1002.4 1131 1631.6 1061.1 973.2 1047.1 1056.5 1179.8 1275 1077.2 876.6 1232.1 726.4 1036.9 1395 978.8

62.9 35.1 51.8 75.2 53 36.6 46.2 44.9 40.9 46.8 44.9 33.6 36.6 28.7 42.2 42.2 43.6

110.5 170.1 56.2 3134.7 3413.4 167.3 859 2687.3 3556.6 127.7 158.8 4966.8 83.6 825.4 94.4 115.4 109.5

1756.7 2476.6 6673.3 2241.7 6335.6 4614.5 8063.2 1734.9 1296.7 9527.5 1715.1 15816.8 1266.7 9123.6 1813.8 5848.1 2223.1

120.9 86.9 97 177.7 123.3 125.6 113.6 143.7 151.6 116.6 160 108.6 86.2 101.6 99.7 129.2 106.7

522.9 427.8 366.5 648.9 634.2 650.4 545.3 475.2 531.7 457 610.4 675.5 492 679.2 485.8 532.1 491.7

14 11.9 10.6 20.8 15.1 14.1 12 13.6 14.4 13.2 13.6 13.3 11.3 14.7 13.5 14.3 12.8

7.7 5.2 5 11.3 5.4 3.9 4.4 6.5 4.7 4.9 6 2.9 4.3 2.9 4.4 4.4 4.4

3.2 3.6 2.9 6.8 2.9 3.5 3.6 4.7 3.3 4.4 4.9 3.5 2.6 2.2 3.1 2.4 3.2

235.3 187.4 152.9 286.5 281 289.9 316.1 215.3 254 186.4 282.3 279.4 196.7 245.1 207.3 247.5 187.2

161.9 123.7 130.3 280.7 183.5 192 159.2 145.6 182.4 143.1 191.7 166.1 130.1 148.5 132.8 161.5 129.5

1164.6 1019.7 862 1580.2 1259.6 1170.1 1088.2 1101.7 1259.6 1017.5 1247.5 1295.4 901.8 1458.4 1104.5 1134 1048.8

698.2 716.7 300.1 1047.8 172.5 789.1 120.7 378.3 493.3 242.3 786.2 1411.9 379.8 1152.9 657.4 481.3 684.9

410.6 334.8 322.1 624.2 479.8 522.5 446.3 409.9 451.2 390.5 522.5 458.5 409.9 453.2 380.9 428.4 349.8

72.2 44.9 60 126.8 69.8 46.6 171.3 66.5 55.9 58.3 101.5 64.1 47.5 32.9 48.3 44.9 38.9

3.6 3.9 3.4 7.9 2.8 2.8 3.5 4.7 4.1 4 4.3 2.9 3 2 3.4 2.3 3.6

1705.1 1336.2 1400.2 2616.4 1603.5 1501.2 1428.3 1542.2 1743.8 1454.5 1921.2 1385.1 1138.4 1594.2 1321 1572 1233.9

950.1 807.3 621.3 1263.3 1104.2 1201.5 1074.8 881.5 923.4 784.5 1030.3 1154 929 1143.2 910.4 935.5 837.5

1597.6 1211.9 1094.8 2213.7 1488 2243.5 1309.8 1323.2 1908 1543 2362 2286.6 1488 1366.5 1269.5 1777.8 1941.7

29.3 24.9 24 42.8 27 20.9 17.7 25.9 26.1 22.8 26.9 20.8 18.8 13.6 22.5 25.5 18.9

402.6 375.3 361.9 558.4 520.5 515 471 388.1 667.7 470.1 500.6 402.8 434.3 303.5 398.9 530.3 321

247.1 202.5 200.9 383.7 306.7 338.2 254.3 300.6 311.8 247.2 316.4 293.2 225.6 309.7 224.7 328.2 236.5

110.9 88.1 88.1 141.7 93.1 86.5 102.6 114.5 112.3 91.5 125.7 81.2 74.8 68 83.9 83 81.2

177.2 151.4 147.7 203.9 168.5 181.7 145 167.9 228.7 237.2 210.6 199.8 152.7 156.9 156.2 174.4 152.4

134.3 118.9 128.2 191.3 122.4 114.8 117.1 119.7 117.1 117.4 120.2 110.7 113.5 106 119.2 118.7 111.8

64.2 56.3 78.9 123.1 64.2 57.3 61.2 72 65.6 64.2 72 56.3 55.3 45.9 59.7 66.6 57.3

488.7 495.4 513.2 634.6 502 527.2 474.9 522.6 573.3 530.2 578.7 604.9 460.6 433.8 570.5 581.4 490.4

1011.5 961.8 1121.3 1239.4 1704.7 2307.7 2234.4 1342.5 2647.9 1424.8 1307 1393.7 1543 1274.3 1114.6 2061.5 874.9

245 205 202.8 311.7 265.9 306 248.7 209.4 283.3 206 266.3 264.1 261.8 310.8 228.5 250.2 194.7

47 33.3 39.5 89.1 55.6 47.3 51.6 46.1 52.4 40.1 62.9 57.8 47.9 51.4 43.4 45.3 46.1

22.9 16.3 15.1 33.2 15.5 16.3 21.6 20.4 15.5 18.8 21.8 15.1 13.5 11.6 15.1 14.3 17.1

Analyte Sample

AC C

EP

TE D

M AN U

SC

RI PT

Samples experiment 1