The optimized anti-CD20 monoclonal antibody ublituximab bypasses natural killer phenotypic features in Waldenstrom macroglobulinemia

The optimized anti-CD20 monoclonal antibody ublituximab bypasses natural killer phenotypic features in Waldenström macroglobulinemia

by Magali Le Garff-Tavernier, Linda Herbi, Christophe De Romeuf, Nabih Azar, DamienRoos-Weil, Patrick Bonnemye, Rémi Urbain, Véronique Leblond, Hélène Merle-Beral, and Vincent Vieillard

Haematologica 2014 [Epub ahead of print]

Citation: Le Garff-Tavernier M, Herbi L, De Romeuf C, Azar N, Roos-Weil D, Bonnemye O, Urbain R, Leblond V, Merle-Beral H, and Vieillard V. The optimized anti-CD20 monoclonal antibody ublituximab bypasses natural killer phenotypic features in Waldenström macroglobulinemia. Haematologica. 2014; 99:xxxdoi:10.3324/haematol.2014.118083

Publisher's Disclaimer.0E-publishing ahead of print is increasingly important for the rapid dissemination of science.Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts thathave completed a regular peer review and have been accepted for publication. E-publishingof this PDF file has been approved by the authors. After having E-published Ahead of Print,manuscripts will then undergo technical and English editing, typesetting, proof correction andbe presented for the authors' final approval; the final version of the manuscript will thenappear in print on a regular issue of the journal. All legal disclaimers that apply to thejournal also pertain to this production process.

Copyright 2014 Ferrata Storti Foundation.Published Ahead of Print on December 31, 2014, as doi:10.3324/haematol.2014.118083.

1

Letter to the Editor

The optimized anti-CD20 monoclonal antibody ublituximab bypasses natural killer

phenotypic features in Waldenström macroglobulinemia

Magali Le Garff-Tavernier1,2,3, Linda Herbi1,2,4, Christophe de Romeuf4, Nabih Azar5,

Damien Roos-Weil1,5, Patrick Bonnemye3, Rémi Urbain4, Véronique Leblond1,5, Hélène

Merle-Beral1,3,6,* and Vincent Vieillard1,2,7,*

1Sorbonne Universités, UPMC, Univ Paris 06, Paris, France; 2INSERM U1135, CIMI-Paris, Paris, France; 3AP-HP, Hôpital Pitié-Salpêtrière, Service d’Hématologie Biologique, Paris, France; 4Laboratoire Français de Fractionnement et des Biotechnologies (LFB), Les Ulis, France; 5AP-HP, Hôpital Pitié-Salpêtrière, Service d’Hématologie Clinique, Paris, France; 6INSERM U1138, Programmed cell death and physiopathology of tumor cells, team 19,

Centre de Recherche des Cordeliers, Paris, France and 7CNRS, ERL8255, CIMI-Paris, Paris, France * HMB and VV contributed equally to this manuscript.

Running heads: Ublituximab bypasses NK features in WM

Correspondence:

Dr Magali Le Garff-Tavernier, Service d’Hématologie Biologique, Groupe Hospitalier Pitié-

Salpêtrière Charles-Foix, Bâtiment de la Pharmacie, 47-83 bd de l’Hôpital, 75651 Paris

Cedex 13, France.

E-mail: [email protected]

Acknowledgments

The authors would like to thank the patients for providing the research samples used in this

study. We also thank C. Blanc and B. Hoareau from the Flow Cytometry Core CyPS (Pierre

et Marie Curie University, Paris, France) for the cell sorting. J. Decocq, M. Brissard, S.

Gueguen and A. Grelier are acknowledged for their technical assistance. This study was

supported in part by funds from the Laboratoire Français de Fractionnement et des

Biotechnologies (LFB, Les Ulis, France), the association la Ligue contre le cancer (RS08/75-

2

4) and INSERM. V. Vieillard is recipient of a Contrat Hospitalier de Recherche

Translationnelle (CHRT).

3

Waldenström macroglobulinemia (WM) is a rare form of lymphoma characterized by

an infiltration of lymphoplasmacytic cells in bone marrow and immunoglobulin M

monoclonal gammopathy.1 Despite the introduction of several therapies, WM remains

incurable.2 Therefore, novel strategies await a better understanding of the mechanisms

underlying the immune surveillance around this pathology. Natural killer (NK) cells were

initially identified through their ability to lyse tumor cells. They express an array of inhibitory,

activating, adhesion and cytokine receptors that enable them to kill targets while sparing

normal cells.3 NK cells also express the low-affinity Fc receptor (FcγRIIIA/CD16), enabling

them to detect antibody-coated target cells and to exert antibody-dependent cell cytotoxicity

(ADCC).3 In this study, we focused our attention on NK cells relative to the presence of a

circulating B-cell clone in WM patients.

The 47 WM patients were diagnosed at the WM French Reference Center Pitié-

Salpêtrière Hospital (Paris, France) according to classical biological criteria.1 This study was

approved by the institutional ethic committee at the Pitié-Salpêtrière Hospital (CPPIDF6) and

all patients provided informed consent prior to participation.

The characteristics of WM patients are summarized in Table 1. They were untreated or

received no treatment during the two years prior to the study, and compared to 20 healthy

blood donors from the Etablissement Français du Sang. In 26/47 patients, circulating B-cell

clone was detected with a frequency ranging from 0.7 to 63.0 % of lymphocytes,

corresponding to 0.1 to 17.0% of leukocytes. NK cells, B- and T-lymphocytes from freshly

harvested whole blood samples were analyzed on the CD45+ lymphocytic gate after staining

with an appropriate antibody cocktail (Supplemental data 1), as previously described.4,5 At

least 100,000 leucocytes were analyzed on a FACSCanto II (BD Biosciences) to assess the

distribution of NK cells, B and T lymphocytes from WM patients, relative to the presence of

circulating B-cells (as defined in Supplemental data 2), and compared with healthy controls.

A similar absolute number of whole CD3+ T and CD19+ B cells in healthy controls and WM

samples are observed (Figure 1a). The absolute value of B cells correlated significantly with

the percentage of a circulating B-cell clone (Figure 1b). The distribution of CD3-CD56+ NK

cells and both CD56dim and CD56bright subsets did not differ between the study groups (Figures

1c-1d), indicating that WM does not disturb this immune subset, compared with other

hematological diseases.5-8

The phenotypic frequency of the CD3-CD56dim cytotoxic NK cell subset was further

investigated on a Gallios (Beckman-Coulter) flow cytometer, while gating out CD3-CD56bright

cell. The frequency of CD56dim NK cells from WM patients was indistinguishable from those

4

of controls in terms of the cell-surface expression of NK receptors (FcγRIIIA/CD16, NKG2A,

NKG2D, NKG2C, ILT-2, LAIR1, DNAM-1, NKp80, and 2B4) (Figure 1e and data not

shown). However, it is important to note that mean of fluorescence intensity (MFI) values for

CD16 are significantly increased in WM patients, whatever the presence of circulating B-cell

clone, as compared to controls (Figures 1e). Additionally, the early activation marker CD69 is

increased in WM, both in absolute value and percentage (Figure 1f), whereas it is decreased in

chronic lymphocytic leukemia (CLL).5 Notably, the expression of each tested receptor

seemed remarkably stable over a 1-year period (Supplemental data 3). More importantly,

CD57, a marker associated with terminal NK differentiation,9 was profoundly increased in the

WM patients, both in percentage and absolute value, and particularly in those with a

circulating B-cell clone (Figure 1g). These data are consistent with those reported by Li et al10

showing that CD57+ cytotoxic T cells are increased in patients with WM. CD57 was also

frequently reported in the association with several cancers, strongly linked to a less severe

disease and a better outcome, in sharp contrast to what was observed for CD8+ T cells.11 We

also observed a weaker expression of the activating NK receptors NKp30 in WM patients,

compared with controls (Figures 1h). In addition, Figure 1h shows an inverse correlation

between CD57 and NKp30 expressions, which is highly more significant in WM patients

(Spearman test: r=-0.91, p<0.0001), than in healthy controls (Spearman test: r=-0.49, p=0.01),

previously reported in the relationship with differentiation of NK cells. Taken together, these

data suggest that CD56dim NK cells in WM are more differentiated in patients with a

circulating B-cell clone. Similar modulations of the NK-cell repertoire was previously

described in the elderly individuals,4 and in patients with acute leukemia,12 but not with CLL.5

We further assessed the overall cytolytic activity of NK cells from WM patients.

Direct cytotoxicity was performed against K562 target cells. NK cells were purified from

fresh peripheral blood mononuclear cells (PBMCs) at the Flow Cytometry Core CyPS (Pitie-

Salpetriere hospital) with a cell-sorting BD FACSAria II flow cytometer. The purification of

CD3-CD56+ NK cells was >98%. Direct cytotoxicity was assessed on purified NK cells

before or after IL-2 stimulation (1,000 U/ml, Proleukin, Chiron) for 48 hours with a standard 51Cr-release assay against the K562 cell line, at a different effector/target (E:T) cell ratio.5,6 A

slight but non-significant decrease of the cytotoxicity mediated by non- or IL2-activated NK

cells was observed in the group of WM patients with a circulating B-cell clone compared with

two other groups (Figure 2a). Of note, granzyme-B and perforin were expressed at equivalent

levels in all samples (Supplemental data 3).

5

ADCC assays were performed with purified non-activated NK cells in the presence of 51Cr-labeled Raji target cells or purified autologous peripheral blood B cells, at an effector

ratio of 1:10 in the presence of anti-CD20 rituximab (Roche Ltd) or ublituximab (LFB

Biotech), an optimized anti-CD20 mAb characterized by a low fucose content in its Fc region

that improves FcγRIIIA (CD16) binding and consequently increases ADCC activity.13 ADCC

against Raji cells was significantly increased in the presence of 1 ng/ml of ublituximab,

compared with rituximab, in all groups (Figure 2b, left panel). However, ADCC against

autologous B cells was significantly increased in WM patients with a circulating B-cell clone

only in the presence of ublituximab, and not of rituximab (Figure 2b, right panel). The

comparison of ADCC mediated in the presence of Raji cells and autologous B-cells revealed

that ADCC efficacy is mainly more dependent of the intrinsic resistance to the tumor target

cells, than on the NK functionality. An additional support for ADCC findings provided from

the degranulation capacities of NK cells, which are highlighted by CD107a cell-surface

expression by flow cytometry, in the presence of these two anti-CD20 mAbs, as described.6

Indeed, regardless of the concentration, ublituximab triggered better effector functions to clear

WM cells in patients with a circulating B-cell clone compared with rituximab (Figure 2c),

which is consistent with findings in CLL patients.5,6 Importantly, the percentage of CD107a+

NK cells significantly correlated with the frequency of circulating B-cell clones only in the

presence of ublituximab (Figure 2d).

Taken together, these data show that the efficacy of NK cells against B-cell clones is

greater with ublituximab than with rituximab, allowing the NK phenotypic features observed

in WM to be bypassed.

Finally, our findings provide additional insights into the cellular mechanisms

underlying the clinical activity of optimized anti-CD20 mAbs. An ongoing phase I/II study of

ublituximab (NCT01647971) to evaluate monotherapy in rituximab-relapse and refractory B-

cell lymphoid malignancies, including WM shows promising early results.14 However, it

should be important to perform side-by-side in vivo experiments in human testing both

ublituximab and rituximab efficacy in WM. In parallel, further combination of optimized anti-

CD20 mAbs with novel chemo-free drugs that target B-cell receptor signaling such as bruton

tyrosine kinase (BTK) inhibitor (ibrutinib) and phosphatidylinositol-3-kinase delta (PI3Kδ)

inhibitor (idelalisib) may achieve complete remission in WM patients.15

6

Authorship

MLT, HMB and VV were the principal investigators and take primary responsibility for the

paper; CdR and RU provided anti-CD20 mAb reagents and participated to the design of the

study; NA, DR, and VL recruited the patients; MLT, LH and PB performed the laboratory

work for this study; MLT, HMB and VV wrote the paper. CdR, NA, DR, RU and VL

critically read and approved the manuscript.

Disclosures

CdR and RU are employed by LFB. Ublituximab was designed, manufactured and provided

by LFB Biotechnologies (Les Ulis, France). CdR has filed patent application

(PCT/FR2005/003123) owned by LFB Biotechnologies covering the use of ublituximab in B-

cell lymphoma. In January 2012, LFB Biotechnologies licensed the exclusive rights to

develop and commercialize ublituximab to TG Therapeutics, Inc. (New York, NY, USA).

MLT, LH, NA, DRW, PB, HMB, and VV have no relevant conflicts of interest to disclose.

References

1. Campo E, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. The 2008 WHO

classification of lymphoid neoplasms and beyond: evolving concepts and practical

applications. Blood. 2011;117(19):5019-32.

2. Leblond V, Johnson S, Chevret S, et al. Results of a randomized trial of chlorambucil

versus fludarabine for patients with untreated Waldenstrom macroglobulinemia, marginal

zone lymphoma, or lymphoplasmacytic lymphoma. J Clin Oncol. 2013;31(3):301-317.

3. Vivier E, Ugolini S, Blaise D, Chabannon C, Brossay L. Targeting natural killer cells and

natural killer T cells in cancer. Nat Rev Immunol. 2012;12(4):239-252.

4. Le Garff-Tavernier M, Beziat V, Decocq J, et al. Human NK cells display major

phenotypic and functional changes over the life span. Aging Cell. 2010; 9(4):527-535.

5. Le Garff-Tavernier M, Decocq J, de Romeuf C, et al. Analysis of CD16+CD56dim NK

cells from CLL patients: evidence supporting a therapeutic strategy with optimized anti-

CD20 monoclonal antibodies. Leukemia. 2011; 25(1):101-109.

6. Le Garff-Tavernier M, Herbi L, de Romeuf C, et al. Antibody-dependent cellular

cytotoxicity of the optimized anti-CD20 monoclonal antibody ublituximab on chronic

lymphocytic leukemia cells with the 17p deletion. Leukemia. 2014;28(1):230–233.

7

7. Farnault L, Sanchez C, Baier C, Le Treut T, Costello RT. Hematological malignancies

escape from NK cell innate immune surveillance: mechanisms and therapeutic

implications. Clin Dev Immunol. 2012;2012:421702.

8. Lion E, Willemen Y, Berneman ZN, Van Tendeloo VF, Smits EL. Natural killer cell

immune escape in acute myeloid leukemia. Leukemia. 2012;26(9):2019-2026.

9. Bjorkstrom NK, Riese P, Heuts F, et al. Expression patterns of NKG2A, KIR, and CD57

define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education.

Blood. 2010;116(19):3853-3864.

10. Li J, Sze DM, Brown RD, et al. Clonal expansions of cytotoxic T cells exist in the blood

of patients with Waldenström macroglobulinemia but exhibit anergic properties and are

eliminated by nucleoside analog therapy. Blood. 2010;115(17):3580-3588.

11. Nielsen CM, White MJ, Goodier MR, Riley EM. Functional Significance of CD57

Expression on Human NK Cells and Relevance to Disease. Front Immunol. 2013;4:422.

12. Fauriat C, Just-Landi S, Mallet F, et al. Deficient expression of NCR in NK cells from

acute myeloid leukemia: Evolution during leukemia treatment and impact of leukemia

cells in NCRdull phenotype induction. Blood. 2007;109(1):323-330.

13. de Romeuf C, Dutertre CA, Le Garff-Tavernier M, et al. Chronic lymphocytic leukaemia

cells are efficiently killed by an anti-CD20 monoclonal antibody selected for improved

engagement of FcgammaRIIIA/CD16. Br J Haematol. 2008;140(6):635-643.

14. Deng C, Effie Amengual J, Schreeder MT, et al. A phase I dose-escalation trial of

ublituximab (TG-1101), a novel anti-CD20 monoclonal antibody (mAb), for rituximab

relapsed/refractory B-cell lymphoma patients. J Clin Oncol. 2013;31(15):8575.

15. Ghobrial IM. Choice of therapy for patients with Waldenstrom macroglobulinemia. J

Clin Oncol. 2013;31(3):291-293.

8

Table 1: Main characteristics of WM patients

Patient number

Sex Age (Year)

Lymphocytosis

(109/L)

% B-cell clone in

lymphocytes

% B-cell clone in

leucocytes

Previous treatment

End of treatment prior the

study #1 F 87 3599 40.0 15.0 No #2 M 66 2960 24.0 13.0 No #3 M 67 3250 39.0 14.0 No #4 M 81 2140 10.0 2.5 No #5 F 69 3115 35.0 10.0 Chloranbucil 15 years #6 F 55 2808 2.5 0.9 No #7 F 77 1744 10.0 2.5 No #8 M 71 Not done 4.0 1.2 No #9 M 59 1476 28.0 4.5 No #10 M 65 1807 3.6 1.0 No #11 M 73 3339 2.8 0.7 No #12 M 49 1174 2.8 1.0 No #13 F 62 2088 3.5 0.8 No #14 F 56 3428 10.0 4.0 Chloranbucil 3 years #15 M 45 2792 4.5 2.0 No #16 M 78 3550 63.0 17.0 No #17 M 61 710 1.8 0.3 No #18 M 67 2200 0.7 0.3 No #19 F 49 1940 10.0 4.0 Rituximab 4 Years #20 M 57 1118 12.0 2.0 Chloranbucil

Fludarabin 13 months

#21 F 63 1480 1.0 0.3 No #22 M 67 1400 26.0 8.0 No #23 M 65 940 1.2 0.1 No #24 M 69 2070 4.0 0.5 No #25 M 35 2027 3.5 1.0 No #26 F 75 620 0.7 0.3 No

#27 F 74 3550 0 0 Rituximab 3 years #28 M 79 2130 0 0 Chloranbucil

Steroids 2 years

#29 M 56 1005 0 0 #30 M 63 1114 0 0 #31 M 56 1693 0 0 #32 M 70 4070 0 0 Chloranbucil

CTX Fludarabin

7 years

#33 M 54 1117 0 0 #34 M 65 Not done 0 0 #35 M 69 1270 0 0 #36 M 83 870 0 0 Fludarabin

Bortezimib 14 months

#37 M 79 2174 0 0 #38 F 49 1591 0 0 #39 M 79 1790 0 0 #40 F 62 3090 0 0

9

#41 M 79 690 0 0 #42 M 67 1540 0 0 #43 F 65 3964 0 0 #44 M 73 1495 0 0 #45 M 47 1609 0 0 #46 F 73 2243 0 0 #47 F 65 1565 0 0 Chloranbucil 3 years

F: Female ; M: Male ; CTX: Cyclophosphamide

10

Figure Legends

Figure 1. Distribution of lymphocyte subsets and characteristics of NK cells from WM

patients with or without circulating B-cell clones. (a) Absolute values of CD3+ T and CD19+

B cells from peripheral blood. (b) Linear regression between absolute value of CD19+ B cells

and the percentage of circulating B-cell clones (WM clone). (c) Absolute value and frequency

of CD3-CD56+ NK cells from peripheral blood. (d) Expression of CD56bright gated on CD3-

CD56+ NK cells. (e) Frequency and mean fluorescence intensity (MFI) of CD16 on CD3-

CD56dim NK cells. (f) Frequency and absolute value of CD69 on CD3-CD56dim NK cells. (g)

Frequency and absolute value of CD57 on CD3-CD56dim NK cells. (h) Frequency of NKp30

on CD3-CD56dim NK cells. Cells were collected from healthy donors (Ctl; circles) and WM

patients without (clone (-); squares) or with (clone (+); triangles) circulating B-cell clones

(WM clone). Horizontal bars represent the median values. Intergroup comparisons were

assessed with the Kruskal Wallis test and Dunn post-test; *P<0.05, **P<0.001, ***P<0.0001.

(i) Linear regression between CD57 and NKp30 frequencies on CD3-CD56dim NK cells in

healthy donors (Ctl, upper panel) and WM patients (lower panel).

Figure 2. Cytolytic activities of NK cells from WM patients with or without circulating B-

cell clones. (a) Direct lysis of non-activated (left panel) and IL2-activated (right panel) NK

cells from healthy donors (Ctl; circles; n=6) and WM patients without (clone (-); squares;

n=4) or with (clone (+); triangles; n=3) circulating B-cell clones. Cytotoxicity was measured

using standard 51Cr release-assays against K562 target cells at different effector:target cells

(E:T) ratio. The mean percentage values ± s.d. (vertical bars) are shown. (b) Comparison of

NK cell ADCC in the presence of rituximab (open symbols) or ublituximab (closed symbols)

against Raji cells at 1 ng/ml of anti-CD20 (left panel) or against purified peripheral blood

autologous B cells at 100 ng/ml of anti-CD20 (right panel). Assays were performed with

purified NK cells from healthy donors (Ctl; circles) and from WM patients without (clone (-);

squares) or with (clone (+); triangles) circulating B-cell clones (WM clone) (E:T ratio at 1:10).

(c) Degranulation response determined using CD107a expression on CD3-CD56+ NK cells in

the presence of 10 ng/ml (left panel) or 1,000 ng/ml (right panel) of rituximab (open symbols)

or ublituximab (closed symbols). Assays were performed on fresh PBMCs from healthy

donors (Ctl; circles) or WM patients without (clone (-); squares) or with (clone (+); triangles)

circulating B-cell clones (WM clone). P-values were calculated with the Wilcoxon rank-sum

test and refer to the comparison between rituximab and ublituximab; *P<0.05, **P<0.001,

11

***P<0.0001. (d) Linear regression between the percentage of CD107a expression on CD3-

CD56+ NK cells and the percentage of circulating B-cell clones (WM clone) after treatment

with 10 ng/ml (left panel) or 1,000 ng/ml (right panel) of ublituximab. All data relative to

ADCC and CD107a expression are presented after subtraction of the values obtained in the

absence of any antibody.

Antibodies  Clone #  Fluorochrome  Compagny NK Cell-surface staining 

Anti-CD3  UCHT1  ECD  Beckman Coulter Anti-CD8  T8  FITC  Beckman Coulter 

Anti-CD16  3G8  FITC  Beckman Coulter Anti-CD56  N901  PC7  Beckman Coulter Anti-CD69  TPI.55.3  PE  Beckman Coulter 

Anti-CD159a/NKG2A  Z199  APC  Beckman Coulter Anti-NKG2D  ON72  APC  Beckman Coulter 

Anti-CD335/NKp46  BAB281  PE  Beckman Coulter Anti-ILT‐2/CD85j  GHI/75  PE  Beckman Coulter 

Anti-CD19  SJ25C1  PerCP Cy5.5  BD Biosciences Anti-DNAM‐1  DX11  FITC  BD Biosciences Anti-LAIR1  DX26  FITC  BD Biosciences 

Anti-2B4  C1.7  PE  BD Biosciences Anti-CD57  HNK‐1  FITC  BD Biosciences 

Anti-CD107a  H4A3  FITC  BD Biosciences Anti-NKG2C  134591  PE  R&D systems 

Anti-NKp80/KLFR1  239127  PE  R&D systems Anti-CD337/NKp30  AF29‐4D12  APC  Miltenyi Biotec 

Intra-cellular staining Anti-Perforin  δG9  FITC  BD Biosciences 

Anti-Granzyme-B  GB11  FITC  BD Biosciences B Cell-surface staining 

Anti-CD19  J3‐119  PC5.5  Beckman Coulter Anti-CD20  B9H9 (HRC20)  PB  Beckman Coulter Anti-FMC7  FMC7  FITC  Beckman Coulter Anti-CD10  ALB1  AF750  Beckman Coulter Anti-CD45  HI30  HV500  BD Biosciences Anti-CD5  L17F12  PE-Cy7  BD Biosciences 

Anti-CD23  EBVCS‐5  APC  BD Biosciences Anti-CD79b  SN8  APC  BD Biosciences Anti-CD38  HB7  PE  BD Biosciences 

Anti Kappa/Lambda  polyclonal  FITC/PE  Dako 

Supplemental data 1 : Antibodies used in this study

+ 

SSC

-A

SSC

-A

CD45-HV500 FSC-A CD19-PC5.5

SSC

-A

Lambda-PE CD79b-APC CD79b-APC CD5-PE.Cy7

Kap

pa-F

ITC

CD

19-P

C5.

5

CD

5-PE

-Cy7

CD

20-P

B

All events Leucocytes Leucocytes

CD19+ Lymphocytes CD19+ Lymphocytes CD19+ Lymphocytes CD19+ Lymphocytes

b

a C

D16

-FIT

C

0 102

103

104

105

0

102

103

104

105

280934.fcs!<PE-Cy7-A>, <APC-A> subset

<APC-A>: CD56

<F

ITC

-A>

: C

D57

0 102

103

104

105

0

102

103

104

105

280936.fcs!<FITC-A>, <APC-A> subset

<APC-A>: CD56

<P

E-A

>: C

D69

0 102

103

104

105

0

102

103

104

105

280952.fcs!<FITC-A>, <APC-A> subset

<APC-A>: CD56

<P

E-A

>: C

D16

0 102

103

104

105

0

102

103

104

105

280934.fcs!FSC-H, SSC-A subset

<PE-Cy7-A>: CD3

<A

PC

-A>

: C

D56

0 50K 100K 150K 200K 250K

0

50K

100K

150K

200K

250K

280952.fcs

FSC-H

SSC-A

0

50

100

150

200

250

150 200 250 100 50 0

FSC-A

SSC

-A

All events Leucocytes

0

CD

56-A

PC

102

103

104

105

0 102 103 104 105

CD3-PE-Cy7

0 102 103 104 105 0 102 103 104 105 0 102 103 104 105

0

102

103

104

105

0

102

103

104

105

0

102

103

104

105

CD56-APC

CD

69-P

E

CD56-APC CD56-APC

CD

57-F

ITC

CD3-CD56dim CD3-CD56dim CD3-CD56dim

Supplemental data 2. Determina,on of a B‐cell circula,ng clone in WM. Lymphocytes were isolated from peripheral blood samples by ga,ng on the forward (FSC)/side (SSC) scaEers and CD45/SSC. (a) The CD19+ B‐lymphocytes were evaluated for the B‐cell surface markers (Supplemental data 1) for the determina,on of the Matutes/Moreau score, which was less than or equal to 3 for all pa,ents. In this representa,ve case (pa,ent #7, Table 1), the B‐cell circula,ng clone, shown in red, was Kappa+CD5+CD79b+CD20+. This B‐cell clone corresponded to 50% of the peripheral B‐lymphocytes and to 10% of the total lymphocytes. Residual normal B lymphocytes are shown in green. (b). The NK cells were  evaluated  on  the  CD3‐CD56+  lymphocy,c  gate  for  the  determina,on  of  a  panel  of  NK‐cell markers (Supplemental data 1), like CD16, CD69 and CD57. 

0

25

50

75

100

0

20

40

60

80

% C

D57

+ 0

10 20

40 50

% C

D69

+

30

% C

D16

+

0

25

50

75

100

% P

erfo

rin+

0

25

50

75

100

% G

ranz

yme

B+

b

a

clone (+) clone (-)

T0 T1 T0 T1 clone (+) clone (-)

T0 T1 T0 T1 clone (+) clone (-)

T0 T1 T0 T1

Ctl clone (-) clone (+)

WM

Ctl clone (-) clone (+)

WM

Supplemental data 3. Stability of CD16, CD57, and CD69 surface expression and intracellular staining of ly,c granule components in NK cells from WM pa,ents with or without circula,ng clones. (a) Cell‐surface markers were tested at To, corresponding to the first  inclusion, and then 12 to 24 months later  (T1)  on NK  cells  from WM pa,ents without  (clone  (‐);  squares)  or with  (clone  (+);  triangles) circula,ng B  clones.  (b)  Comparison of  intra‐cellular  expression of perforin  and granzyme‐B  in NK cells collected from healthy donors (Ctl) and WM pa,ents without (clone (‐); squares) or with (clone (+); triangles) circula,ng B clones. Horizontal bars represent the median value.