Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Nair S, Branagan AR, Liu J, Boddupalli CS, Mistry PK, Dhodapkar MV. Clonal immunoglobulin against lysolipids in the origin of myeloma. N Engl J Med 2016;374: DOI: /NEJMoa

2 Supplementary Appendix Supplement to: Nair S, Branagan AR, Liu J, Boddupalli CS, Mistry PK and Dhodapkar MV. Clonal immunoglobulin against lysolipids in origin of myeloma Contents Supplementary Methods Page 2 Table S1 Page 3 Figure S1-S8 Page 4-11 References Page 12 1

3 Supplementary Methods: Protein electrophoresis (SPEP) and Immunofixation (IFE): Protein electrophoresis was carried out in agarose gels using a Quick gel chamber (Helena Laboratories) according to the manufacturer s instructions. Some samples were also subjected to immunofixation using anti-sera directed against human heavy (α, γ and μ) and light (κ and λ) immunoglobulin chains. IgG and F(ab')2 purification from lipid reactive MG patient plasma: IgG from plasma of lipid reactive MG patients was purified by affinity chromatography using HiTrap Protein G columns (GE health life sciences). The purification of the monoclonal F(ab')2 was obtained by using Pierce F(ab')2 Preparation Kit. Protein concentration was checked using BCA protein assay kit (Pierce), and the antibody purity was assessed in both non-denaturing and denaturing electrophoresis (sodium dodecyl sulfate-8-12% polyacrylamide gel electrophoresis [SDS-8-12% PAGE]) assay. The purity was further confirmed by performing western blot using anti-f(ab')2 and anti-igg HRP respectively. Lipid binding affinities of F(ab')2 fragment of lipid reactive IgG were estimated by performing ELISA based lipid recognition and immunoprecipitation with sphingosine coated beads (Echelon Biosciences). Affinity measurement using ELISA: The dissociation constant (K d) of lipid-antibody interactions was determined by ELISA as described 1. Briefly double sandwich ELISAs were performed with sequential incubation of increasing concentration of biotinylated purified IgG from plasma of lipid reactive MG patient (20-200nM) and fixed LGL1 concentration (1nM) in microtiterplates coated with purified lipid reactive IgG at 1-5 μg/ml, following which the bound antibody was indirectly revealed with using anti-streptavidin peroxidase. A plot of the optical density versus the concentration of antibody was fitted using a regression program, to a hyperbola fitting using the Michaelis Menten equation and used to derive values for K d. 2

4 Table S1. Clinical characteristics of patients with monoclonal gammopathy with or without lipidreactivity of clonal Ig. Characteristic Lipid Reactive Patients Non-Lipid Reactive Patients P value All patients (N=66) 22 (33%) 44 (67%) Median age Male 15 (68%) 24 (55%) 0.42 MGUS / AMM 5 (23%) 11 (25%) 0.9 Myeloma 17 (77%) 33 (75%) Heavy chain type: IGG 18 (82%) 36 (82%) 0.9 Heavy chain type IGA 3 (14%) 7 (16%) Light chain only: 1 (4%) 1 (2%) Light chain type: kappa 20 (91%) 28 (64%) 0.03 ISS staging at diagnosis (N=41) ISS stage I 7 (58%) 7 (24%) ISS stage II/III 5 (42%) 22 (76%) 0.06 Cytogenetic analysis Ŧ (N=46) Standard Risk cytogenetics 13 (87%) 17 (55%) Intermediate Risk cytogenetics 2 (13%) 11 (35%) High Risk cytogenetics 0 3 (10%) 0.04 Abbreviations: AMM, asymptomatic multiple myeloma; ISS, International Staging System; MGUS, monoclonal gammopathy of undetermined significance. Number of patients with available data regarding the corresponding clinical characteristic. Ŧ Risk based on cytogenetic and FISH mutations using the Stratification for Myeloma and Risk-adapted Therapy (msmart) algorithm, Mikhael et al. Mayo Clin Proc 2013;88: v12. 3

5 Fig S1 WT PBS PBS GBA1 -/- mice LGL1 PBS GBA1 -/- mice LGL CD19 B CD19 CD Spleen BM GL7 Fas CD138 CD38 Figure S1. Gating strategy for GC B cells and plasma cells. Representative contour plots showing the percentage of FAS + GL-7 + germinal center (GC) B cells among total B cells (CD19 + B220 + ) in WT and GBA-/- mice splenocytes 7 days after 3 weeks injection of either PBS or LGL1 immunization (left panel). Representative FACS plot showing the expression of CD38 + CD138 + plasma cells among CD45 low CD19 - cells in the bone marrow of GBA-/- mice after PBS or LGL1 immunization (right panel). 4

6 Fig S2 2.0 OD at 450 nm PBS α-hel Ab HEL PBS HEL α-lgl1 Ab Fig S2 Immunization of GBA-/- mice with HEL does not lead to induction of anti-lgl1 antibodies Bar graph showing the presence of anti-hel and anti- LGL1 antibodies in the sera of GBA-/- mice immunized with PBS or HEL. Data are represented as mean ± SEM (n=3). 5

7 Fig S3 Polyclonal gammopathy (non-gd) SPEP LGL1-blot Figure S3. Lipid reactivity in non-gd polyclonal gammopathy patient sera SPEP analysis (upper panel), followed by LGL1 specific blotting (lower panel) on sera from non-gd polyclonal gammopathy patients. 6

8 Fig S4 Purified IgG DAG CA Lipid A LGL1 (OD at 450 nm) Sporadic MG Healthy Donor Fig S4 Specificity of clonal Igs reactivity against LGL1 Bar graph showing the presence of anti-diacylglycerol (DAG), Cardiolipin (CA), Lipid A and LGL1 antibodies in equivalent amount of purified IgG from patients with sporadic lipid reactive MG or healthy donors. Data are represented as mean ± SEM. 7

9 Fig S PT with Ig M spike PT with Ig M spike α-lipid Ab α-lipid Ab 1.2 OD at 450 nm P P P P020 P037 P044 Lipid reactive MG sera Lipid non- reactive MG sera Figure S5. Light chain skewing of anti-lgl1 antibodies from sera of MG patients displaying lipid reactivity and those lacking lipid reactivity Diluted patient sera (1:250) was added to plate s pre-incubated with LGL1 (500 ng/ml) and probed with α-ig or Ig HRP. Samples were measured in duplicate. Data are representative of mean+ SEM. 8

10 Fig S6 P021(IgG P023 (IgA 606 (IgG 631(IgG i SPEP ii α-ig - HRP LGL1-blot iii α-ig - HRP iv α-iga HRP v α-iga+m+g ( + )- HRP Figure S6. Probing LGL1 blots with anti-igg/igl antibodies SPEP gel showing M spikes from the sera of lipid reactive MG patients (i), LGL1-specific blotting was performed on sera of lipid reactive MG patients and probed with α-ig HRP (ii) or with α-ig HRP (iii) or with α-iga HRP (iv) or with α-iga+m+g ( + ) HRP (v). The IgH/IgL reactivity of clonal Ig for each patient is noted in parentheses. 9

11 Fig S7 0.8 Anti-lipid Ab (OD at 450nm) F(ab )2 IgG Ligand dependent enrichment of clonal Ig C S α-human F(ab ) 2 HRP Figure S7. Lipid- reactivity of F(ab )2 fragments Top panel: Lipid reactivity of F(ab )2 fragment is compared with intact IgG. Equimolar concentration of purified IgG or F(ab )2 fragment were added to plate s pre incubated with LGL1 (500ng/ml) and probed with either anti-igg or F(ab )2 HRP. Samples were measured in duplicates. Data are represented as mean ± SEM. Bottom Panel: Ligand dependent enrichment of clonal Ig by F(ab )2 fragment: Purified F(ab )2 fragments were incubated with control (C) or sphingosine (S) coated sepharose beads;and bead binding fraction was analyzed for the presence of clonal Ig by western blot. 10

12 Fig S8 Absorbance (450 nm) g/ml 3 g/ml 2 g/ml 1 g/ml K d ~ x 10-9 M Antibody concentration (nm) Figure S8. Estimation of affinity of antibody using ELISA A plot of the ELISA signal versus the concentration of lipid reactive antibody derived from simultaneous incubations of increasing concentration of biotinylated purified IgG from the plasma of lipid reactive MG patient ( nm) and fixed LGL1 concentration (1nM) in microtiterplates coated with purified lipid reactive IgG at 1-5 g/ml, following which the bound antibody was indirectly revealed by using anti-streptavidin peroxidase. The plot is then fitted, using a regression program, to a hyperbola. Data were analyzed using the Michaelis Menten equation and used to calculate K d. 11

13 References: 1. Hoylaerts MF, Bollen A, De Broe ME. The application of enzyme kinetics to the determination of dissociation constants for antigen-antibody interactions in solution. J Immunol Methods 1990;126: