1. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY TNFRSF13B/TACI and TNFRSF13C/BAFFR in B cell chronic lymphocytic leukemia

2. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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

3. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY Novak et al, Blood 2002 Hait 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

4. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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

5. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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

6. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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

7. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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). 2.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. 3.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

8. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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.

9. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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

10. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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 (%)

11. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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 PtsDiseaseTACI (%)Hypermutation status(%) CD38 (%) 1.B-CLL0.199.65.4 2.B-NHL1.198.23.6 3.B-CLL2.299.673 4.B-CLL2.290.11.4 5.B-CLL2.598.823.2 6.B-CLL16.9951 7.B-CLL21.99618.5 8.B-CLL29.899.663.8 9.B-CLL43.594.81.9 10.B-CLL54.396.20.3 11.B-CLL78.599.624.1 12.12.B-NHL84886 Low TACI Medium TACI High TACI hypermutation status ≤ 98% good prognosis CD38 > 20% bad prognosis

12. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY CONTROLBAFFAPRIL CONTROLBAFF TACI protects B-CLL cells from apoptosis TACI=2.1% TACI=50.1% aliveEarly apoptotic Late apoptotic&dead aliveEarly apoptotic Late apoptotic&dead aliveEarly apoptotic Late apoptotic&dead aliveEarly apoptotic Late apoptotic&dead aliveEarly apoptotic Late apoptotic&dead aliveEarly apoptotic Late apoptotic&dead Results - 4

13. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY B-CLL NHL Healthy 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 Normal B cells At the relapse of the disease Results - 5

14. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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 patients0 (0 – 55.0)5.11 (0.80 - 64.06) NHL patients 9.0 (0 – 152.0)2.21 (0 - 5.11) Healthy donors49.5 (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

15. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY 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.

16. D EPARTMENT OF I MMUNOLOGY & H ISTOCOMPATIBILITY – U NIVERSITY OF T HESSALY THANK YOU