d epartment of i mmunology & h istocompatibility – u niversity of t hessaly tnfrsf13b/taci and...

DEPARTMENT OF IMMUNOLOGY & HISTOCOMPATIBILITY – UNIVERSITY OF THESSALY

TNFRSF13B/TACI and TNFRSF13C/BAFFR in B cell chronic lymphocytic leukemia


Introduction

TACI:

• Receptor of BAFF and APRIL

participates in:

• Survival and differentiation of B cells

• T cell-independent immune respon-ses

• Antibody production

• Isotype switching

• Homeostasis of B cells

BAFF-R:

• Receptor of BAFF

• Expressed on naïve B cells triggering their survival and maturation

Mackay et al, Cytokines & Growth Factor Reviews 2008


Novak et al, Blood 2002Hait et al., Immunology 2006

Jian et al., Medical Journal 2008

TACI and BAFFR might participate in the survival of B-CLL

cells, protecting them from apoptosis through NFκΒ

activation

Introduction


Anti-TACI, anti-BAFF and anti-APRIL therapeutic

approaches are now available, for the treatment of

patients with autoimmune diseases and might be useful

in patients with B-CLL • Phase I/II study of TACI-Ig to neutralize APRIL and BlyS (R) in patients with refractory or relapsed multiple myeloma or active previously-treated Waldenstrom’s macroglobuliemia.

Rossi et al, Blood 2005

• Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells.

Lee et al, Blood 2006

• Phase I clinical study of Atacicept in patients with relapsed and refractory B-cell Non-Hodgkin’s Lymphoma. Ansell et al, Clin Cancer Res, 2008

• Ib trial of Atacicept, a recombinant protein binding BlyS and APRIL, in patients with chronic lymphocytic leukemia (B-CLL) Kofler et al, Leukemia 2011

• Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas.

Guadagnoli et al, Blood 2011

Introduction


The determination of TNFRSF13B/ TACI and

TNFRSF13C/ BAFFR expression on B-CLL cells and

their contribution to the phenotype and the

prognosis of the disease

Aim of the study


Bone marrow

104 patients with B-CLL (M/F: 59/45, mean age: 68.5 y)

30 patients with low-grade NHL

(M/F: 18/12, mean age: 77.3 y) &

145 healthy donors (M/F: 84/61, mean age: 67.8 y)

Peripheral blood

Materials & Methods


1. Hematologic, serologic and flow cytometric

analyses (for diagnostic purposes), including in

B-CLL a further analysis of the most powerful

prognostic factors (ZAP-70, CD38 and IgH

mutational status).

1. TACI and BAFFR protein expression was detected

by flow cytometry using monoclonal

antibodies (TACI clone 1a1, Abcam and BAFFR

clone 11C1 Biolegend, respectively) in all

samples.

2. TNFRSF13B and TNFRSF13C mRNA expression were

estimated by qRT-PCR in 29 samples (19 with B-

CLL, 8 with NHL and isolated B cells from 2

healthy donors).

Materials & Methods


Materials & Methods

4. B-CLL cells exhibiting high or absent TACI, were

stimulated by PKW (pokweed) 2.5 ng/mL, BAFF 1

μg/mL and APRIL 200 ng/mL and the apoptosis

status was estimated by flow cytometry using an

Annexin V-FITC/ 7-AAD (7-AAD) kit.

5. BAFF and APRIL serum levels were estimated by

ELISA in 37 samples (16 with B-CLL, 11 with NHL

and 10 healthy donors).

6. The statistical analysis was performed using the

SPSS software ver.16.0.


Results - 1

TACI=2.1% TACI=55% TACI=90.1%

Variable TACI mRNA and protein expression on B-CLL cells

BAFFR protein expression

B-CLL cells normal B cells

MFI=6 MFI=11


Results - 2

TACI=2.1% TACI=55% TACI=90.1%

Variable TACI mRNA and protein expression on B-CLL cells

TACI mean expression ± STDEV

B-CLL patients

13.4 ± 22% p < 0.05

NHL patients 36.8 ± 29% p = 0.340

Healthy donors

27.6 ± 14.2%B-CLL NHL Healthy

TACI

expression (%)


TACI expression was not

asso-ciated with

(p>0.05) :

•the presence of

autoimmunity,

•hypogammaglobulinemia,

•monoclonal gammapathy,

•the infection risk and

•the known prognostic

factors of B-CLL

Results - 3

Pts Disease TACI (%)

Hypermutation

status(%)

CD38 (%)

1. B-CLL 0.1 99.6 5.4

2. B-NHL 1.1 98.2 3.6

3. B-CLL 2.2 99.6 73

4. B-CLL 2.2 90.1 1.4

5. B-CLL 2.5 98.8 23.2

6. B-CLL 16.9 95 1

7. B-CLL 21.9 96 18.5

8. B-CLL 29.8 99.6 63.8

9. B-CLL 43.5 94.8 1.9

10. B-CLL 54.3 96.2 0.3

11. B-CLL 78.5 99.6 24.1

12. B-NHL 84 88 6

Low TACI

Medium

TACI

High TACI

hypermutation status ≤ 98% good prognosis CD38 > 20%

bad prognosis


CONTROL BAFF APRIL

APRILCONTROL BAFF

TACI protects B-CLL cells from apoptosis

TACI=2.1%

TACI=50.1%

alive Early apoptotic

Late apoptotic&dead


Late apoptotic&dead


Late apoptotic&dead


Late apoptotic&dead


Late apoptotic&dead


Late apoptotic&dead

Results - 4


B-CLL NHL Healthy

BAFFR_MFI

BAFFR_MFI Mean ± STDEV

B-CLL patients

6.71 ± 2.9% p < 0.05

NHL patients 6.7 ± 3.7% p = 0.05

Healthy donors

10.7 ± 5.9%

BCLL cells

BCLL cells

Normal B cells

At the relapse of the disease

Results - 5


Serum BAFF was low and APRIL high in the majority of B-CLL

patients compared to NHL patients and healthy donors

BAFF pg/ml Median (Range)

APRIL pg/ml Median (Range)

B-CLL patients

0 (0 – 55.0) 5.11 (0.80 - 64.06)

NHL patients 9.0 (0 – 152.0) 2.21 (0 - 5.11)

Healthy donors

49.5 (21.8 – 92.0) 4.78 (2.47 - 18.55)

Soluble BAFF levels was low to undetectable in B-CLL patients. Kreuzaler et al, J Immunol

2012

APRIL serum levels predict time to first treatment in patients affected by B- cell chronic lymphocytic leukemia. Tecchio et al, Eur. J. Haematol 2011

Results - 6


Conclusions

TACI expression is:

• low in the majority of B-CLL patients

• not associated with disease prognosis

• accompanied by very low or absent serum BAFF and high serum APRIL levels

BAFFR MFI expression is:

• low in the B-CLL and NHL patients

These findings should be taken into account in the

case of anti-TACI and anti-BAFFR therapeutic

approaches.


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d epartment of i mmunology & h istocompatibility – u niversity of t hessaly tnfrsf13b/taci and...

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