FDC-Specific Functions of p55tnfr and IKK2

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1 Supplemental Data FDC-Specific Functions of p55tnfr and IKK2 in the Development of FDC Networks and of Antibody Responses Panayiotis Victoratos, Jacques Lagnel, Sotiria Tzima, Marat B. Alimzhanov, Klaus Rajewsky, Manolis Pasparakis, and George Kollias Supplemental Experimental Procedures Semiquantitative RT-PCR and Real-Time PCR Total RNA was extracted using TRIZOL reagent (Invitrogen) according to the manufacturer s specifications. Reverse transcription was performed with 5 µg of total RNA, 0.5 ng of oligo-dt, 10 mm DTT, 1x BSA, 0.25 mm dntps, and 200 units of reverse transcriptase (Promega) in a 20 µl final volume for 1 hr at 37 o C. For semiquantitative RT-PCR, serial dilutions of reverse transcription reactions were amplified with a PTC-200 peltier thermal cycler (MJ Research) in a 20 µl final volume containing 0.5 µm of the following primers: CXCL13 (5 -AGA TCG GAT TCA AGT TAC GCC C-3 and 5 -GCC TGG ACC TTT AAG CTG AGG A-3 ); CCL19 (5 -TTC ACG CCA CAG GAG GAC ATC T-3 and 5 -CCA CAC TCA CAT CGA CTC TCT AGG C-3 ); CCL21 (5 -TGA GCC TCC TTA GCC TGG TCC T-3 and 5 -CGC CAT GGA GAG CAG GTT CA-3 ); VCAM-1 (5 -AAC GAG GCT GGA ATT AGC AGA A-3 and 5 -CCC TCT TTG ACA CTC TTA GAT GGA A- 3 ); ICAM-1 (5 -AGT CCG CTG TGC TTT GAG AA-3 and 5 -TCC TTG CCT ACT TGC TGC CA-3 ); GAPDH (5 -TCT TCT TGT GCA GTG CC-3 and 5 -ACT CCA CGA CAT ACT CAG C-3 ). PCR conditions were optimized for each pair of primers. Amplified products were subjected to agarose gel electrophoresis and subsequent quantification with GelWorks 1D Advanced software (NonLinear Dynamics Ltd). GAPDH was measured in all samples as endogenous control and gene expression levels were corrected for differences in GAPDH content. For real-time PCR, cdna equivalent to 10 ng of total RNA was used per PCR reaction. cdna from 40 ng of RNA derived from one of the wt recipient mice was used as relative calibrator cdna in 1:4 serial dilutions. Amplification conditions were 95 C for 10 min, followed by 40 cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for 30 s. PCR primers have been previously described (Shaffer et al., 2002). Each PCR was performed in duplicate with the iq SYBR Green Supermix (Bio-Rad Laboratories) in an icycler iq real-time PCR detection system (Bio-Rad Laboratories), and the PCR results were analyzed on the icycler Software. Transcript quantity was calculated using the comparative C T method, normalized to GAPDH, and reported as n-fold difference relative to the calibrator cdna. The melting curve profile validated the absence of primer-dimer formation for each oligonucleotide set and the amplification of one product. Supplemental Reference Shaffer, A.L., Lin, K.I., Kuo, T.C., Yu, X., Hurt, E.M., Rosenwald, A., Giltnane, J.M., Yang, L., Zhao, H., Calame, K., and Staudt, L.M. (2002). Blimp-1 orchestrates

2 plasma cell differentiation by extinguishing the mature B cell gene expression program. Immunity 17, Figure S1. CD21-Cre Induces Recombination Specifically in B Lymphocytes and FDCs GFP expression following Cre recombination was measured by FACS analysis of splenocytes from CD21-Cre Z/EGFP +/- mice in comparison with wt (negative control) and Z/EG +/- (complete recombined control) mice (5 mice per group). Analysis of FDCs was performed in partially purified preparations from a pool of 10 mice per group by MACS column with a FS-SS gate on FDCs. Numbers indicate the percentage of recombination as the number of GFP + cell subset per total number of the examined cell subset.

3 Figure S2. Additional Characterization of p55tnfr Mutant Mouse (A) Measurement of p55tnfr expression on CD11b hi splenocytes from indicated groups of mice by FACS analysis shows that the p55tnfr cneo allele is hypomorphic. (B) Tissue specificity of p55tnfr activation. DNA isolated from various tissues of a CD21-Cre p55tnfr neo/- mouse was subjected to Southern blot analysis after digestion with BamHI. Deletion is detected in DNA from spleen and lymph nodes. (C) Purification process of splenic FDCs and the respective purity of each step. (D and E) Cytospin preparations of isolated FDCs were stained with hematoxylin/eosin (original magnification x400) (D) or with CD35 (CR1) antibody (original magnification x800) (E). (F) p55tnfr reexpression after Cre recombination was measured by FACS analysis of MACS column purified FDCs from CD21-Cre p55tnfr cneo/- mice in comparison with wt and p55tnfr D/- mice.

4 Figure S3. Efficient Cre-Mediated Recombination in Ikk2 FL/FL Mice (A) Efficient Cre-mediated recombination was detected by PCR analysis of DNA from isolated FDCderived of CD21-Cre p55tnfr FL/FL mice in comparison with tail DNA of the indicated control mice. (B) Immunofluorecence for RelA and FDC-specific marker in untreated or hutnf-α stimulated (10 ng ml -1 for 30 min) mutant and control FDCs that were purified partially by MACS column.

5 Figure S4. Bone Marrow Transfer from Wild-Type Donors Results in Equal Reconstitution of Ikk2 FDCwt and Ikk2 FDCko Mice (A) Three months after BM transfer, splenocytes from the indicated groups (5 mice per group) were examined for haematopoietic cell repopulation by FACS analysis and (B) the data were expressed as mean frequencies ± SE.

6 Figure S5. Semiquantitative RT-PCR on GC-B Lymphocytes (A) GC-B lymphocytes (B220 + PNA + ) were isolated from spleens of Ikk2 FDCwt and Ikk2 FDCko mice 5 days postsecondary immunization with SRBC by FACS sorting. (B) Total RNAs from GC-B lymphocytes (3 mice per group) were examined for expression of the indicated transcription factors by semiquantitative RT-PCR in 4-fold serial dilutions.

7 Figure S6. FDCs from Ikk2 FDCwt and Ikk2 FDCko Mice Have Similar Efficiency in IC Trapping Mice were passively immunized with preformed PAP ICs, and 24 hr later, ICs were identified by their peroxidase activity in cryostat splenic sections. Mice immunized with a rabbit IgG serum were used as negative controls.

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