A collection of 31 splenic specimens from patients (42.4±17.7 years old) with ITP

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1 Supplemental Materials & Methods Patients and patient samples. A collection of 31 splenic specimens from patients (42.4±17.7 years old) with ITP requiring splenectomy and 36 splenic specimens obtained from subjects undergoing splenectomy because of trauma (42.0±18.9 years old) were analyzed in this study. Sixty-one (26 from ITP/35 from trauma patients) splenic specimens were treated with formalin fixation before they were embedded in paraffin. In addition, fresh splenic tissues obtained from five patients with ITP undergoing splenectomy and one spleen from a non-autoimmune patient were cut into 1 to 1.5 cm 3 pieces, and each piece was embedded in Tissue-Tek O.C.T. compound (Miles, Naperville, IL, USA) before being snap-frozen in liquid nitrogen. The embedded spleen pieces were stored at - 80 C for further investigations. The patients characteristics are summarized in table 1 and 2. The study has been approved by the institutional review board at Charité University Hospitals, Berlin, Germany. Quantification of cells in splenic lymphoid nodules Quantification of Ki67 +, CD3 +, PD-1 + and Foxp3 + ells was performed as follows. ITP and control spleen sections were stained with anti-ki67, anti-cd3, anti-foxp3 or anti- PD-1 Abs. All slides were screened using the Zeiss Axio Imager Z1 (Carl Zeiss, Jena, Germany) with a magnification of 200x. 1

2 To evaluate the percentage of proliferating cells in GCs compared to PLNs, the total number of cells and the number of Ki67 + cells were evaluated within both splenic structures using AxioVision software (Carl Zeiss). In control spleens, between 129 to 571 cells in GCs and between 17 to 225 cells in PLNs were counted. In ITP spleens, between 87 to 784 cells in GCs and 22 to 215 cells in PLNs, respectively, were evaluated. Representative sections of spleens from ITP (n=6) and controls (n=6) were stained with anti-cd3 and anti-pd-1 antibodies and counterstained with hematoxylin to reveal the presence of T cells and T FH. The frequencies of CD3 + T cells and PD-1 + cells were enumerated and adjusted to the area of PLNs or GCs since PLNs were significantly smaller (10.5± µm 2 ) than the GCs (52.0± µm 2, p<0.0001). The areas of both structures were quantified using AxioVision software (Carl Zeiss, Jena, Germany). Immunofluorescence staining of frozen spleen samples In order to identify B cell subsets and FDCs, three frozen splenic tissue specimens from patients with ITP were stained and analyzed using dual immunofluorescence. Serial 5-6 µm thick cryostat-sections from spleens were cut and mounted onto SuperFrost Plus slides (Menzel GmbH & Co, Braunschweig, Germany). The sections were fixed in ice cold acetone at 4 C for 5 minutes and dried before further use. 2

3 The slides were initially incubated with the monoclonal antibodies at the appropriate concentration for 40 minutes at RT. After three washing steps with TBS, the slides were incubated with the secondary antibodies either FITC-conjugated goat anti mouse antibody (Dako Deutschland GmbH, Hamburg, Germany) or RRX-conjugated donkey anti mouse antibody (Jackson research, Dianova GmbH, Hamburg, Germany) for 40 minutes at RT, then washed carefully three times for 5 minutes. Afterwards, the slides were incubated for 40 minutes at RT with rabbit polyclonal anti- CD20 (Neomarkers, Fremont, CA, USA) or Rabbit polyclonal anti- IgM (Dako). Again, the slides were washed with TBS and subsequently incubated with either FITC conjugated donkey anti-rabbit antibody or RRX conjugated donkey anti-rabbit antibody (Jackson research). Finally, after three washing steps in TBS, the slides were covered with Vectashield Hard set without or with DAPI (Vector, Burlingame, CA, USA) and subsequently analyzed using a Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss, Jena, Germany). All analyses were carried out on three different specimens and from different areas in each specimen. Rabbit anti-cd20 pab (Neomarkers, Thermo Fisher Scientific GmbH, Dreieich, Germany, 1:200) revealed by Rhodamine Red X (RRX)-conjugated donkey antirabbit Ig antibody (Jackson research, Dianova GmbH, Hamburg, Germany, 1:100) was combined with either fluorescein isothiocyanate (FITC)-conjugated anti-ki67 mab (MiB-1, Dako Deutschland GmbH, 1:20) or with FITC-conjugated anti-cd3 mab (UCHT1, BD Bioscience, Heidelberg, Germany, 1:50) or with FITC-conjugated anti- 3

4 IgM mab (G20-127, BD Bioscience, 1:20) or with anti-cd1c mab (C4445, Lifespan Bioscience, Eching, Germany,1:20) or with FITC-conjugated anti-cd27 mab (M- T271, BD Bioscience,1:20) or with FITC-conjugated anti-igd (IA6-2, BD Bioscience,1:20) or with unconjugated anti-gpiiia/iib mab (2B3A, Enzyme Research Laboratories,1:100) or anti-gpiv (3H1265, Lifespan BioSciences). All unconjugated mouse antibodies as well as FITC-conjugated mouse antibodies were detected with FITC-conjugated goat anti-mouse Ig. The sections were visualized by using Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss, magnification: 200x). FDCs were identified by their expression of CD35 (E11, BD bioscience, 1:20). Unconjugated-rabbit anti-igm pab (Dako, 1:400) or anti-cd20 pab (Neomarkers) was detected with RRX-conjugated donkey anti-rabbit Ig and combined with FITCconjugated anti-cd35 mab or anti-gpiib/iiia mab (2B3A, Enzyme Research Laboratories, South Bend, IN, USA, 1:100) detected with FITC-conjugated goat antimouse Ig antibody (Dako Deutschland GmbH, 1:30). The sections were visualized by Zeiss LSM 710 confocal microscopy (Carl Zeiss; magnification: 200x) and analyzed with Zen 2009 light edition (Carl Zeiss). The proportion of total PLNs to GPIIb/IIIareactive PLN was analyzed by co-staining with anti-igm and anti-gpiib/iiia (2B3A, Enzyme Research Laboratories, 1:100) on 5 frozen spleens. 67 PLNs of 4 ITP spleens and 32 PLNs of one control spleen were screened for the presence of GPIIb/IIIa. 4

5 Supplementary figure. Comparative analyses of the density of lymphoid nodules and germinal centers (GC) within the white pulp of spleens obtained from patients with primary (n=13) and secondary ITP (n=6). There were no significant differences between patients with primary and secondary ITP. 5

6 Supplementary figure.