Phenotypic and Morphological Properties of Germinal Center Dark Zone Cxcl12-Expressing Reticular Cells.

Mice and chimeras C57BL/6 (B6) and B6-CD45.1 mice were obtained from The Jackson Laboratory or the National Cancer Institute. B6.Cg-Cxcl12 tm2Tng ( Cxcl12- GFP) gene-targeted mice were backcrossed to the C57BL/6 background more than 7 generations and provided by T. Nagasawa ( 19 ). Tg(UBC-GFP)30Scha/J (UBI-GFP) transgenic mice were backcrossed to the C57BL/6 background for more than 8 generations and were from The Jackson Laboratory ( 20 ). B6.Tg(Cr2-Cre)3Cgn ( CD21 -Cre) BAC-transgenic mice were fully backcrossed to C57BL/6 and provided by K. Rajewsky (Immune Disease Institute, Boston, MA) ( 21 ). B6.Cg-Gt(ROSA)26Sor tm6(CAG-Zsgreen1)Hze/J ( R26 -ZsGreen) mice have a CAG promoter, a floxed stop sequence and ZsGreen1 knocked into the Gt(ROSA)26Sor locus and were from The Jackson Laboratory. C57BL/6N-Tg(Ccl19-cre) 489Biat ( Ccl19- Cre) BAC-transgenic mice ( 8 ) were provided by C. Lowell. Gt(ROSA)26Sor tm1(EYFP)Cos ( R26 -EYFP) transgenic mice express EYFP from the Gt(ROSA)26Sor locus after Cre-mediated deletion of floxed stop cassette ( 22 ) and were provided by L. Lanier. Tg(CAG-ECFP)CK6Nagy (CFP) transgenic mice were backcrossed to the C57BL/6 background more than 5 generations and were from The Jackson Laboratory. Tg(IghelMD4)4Ccg (MD4) transgenic mice were fully backcrossed to C57BL/6 and were from an internal colony. To make bone marrow (BM) chimeras, UBI-GFP mice were treated intraperitoneally (i.p.) with 500μg anti-Thy1.2 (clone 30H12) before being lethally irradiated and reconstituted for at least 8 weeks with wild-type CD45.1 BM. CD21 -Cre mice were crossed to R26 -ZsGreen mice and lethally irradiated and reconstituted for at least 8 weeks with wild-type CD45.1 BM as described previously ( 15 ). Animals were housed in a specific pathogen–free environment in the Laboratory Animal Research Center at the University of California, San Francisco (UCSF), and all experiments conformed to ethical principles and guidelines approved by the UCSF Institutional Animal Care and Use Committee. Infections and Immunizations Mice were infected with acute LCMV-Armstrong intravenously (i.v.) at 2.5x10 5 pfu and analyzed at day 15 for GCs in pLN and spleen ( 23 ). For induction of spleen GCs, mice were immunized i.p. with 2x10 8 SRBCs (Colorado Serum Company) on day 0 and day 5 and were analyzed on day 10–12. For induction of pLN GCs, animals were immunized subcutaneously (s.c.) at the shoulders, flanks and above the tail with SRBCs on day 0 and day 5. Draining pLNs (axillary, brachial and inguinal) were analyzed on day 10–12. Treatments and Transfers For LTβR and TNFR signaling blockade, Cxcl12- GFP mice were immunized i.p. or s.c. with SRBCs on day 0 and day 5 and on day 10 treated i.v. with 100μl each of 1mg/ml mLTβR-huIgG1 (LTβR-Fc, provided by J. Browning) and 1mg/ml TNFR55-huIgG1 (TNFR-Fc, provided by J. Browning) or saline. Tissues were analyzed 4 days later. For CXCR4 inhibitor treatment, Cxcl12 -GFP mice were immunized with SRBC i.p. on day 0 and day 5. On day 8, Alzet osmotic pumps (1-day duration, 8.4μl/h pumping rate; Model 2001D; Durect Corporation) loaded with saline or 5mg/ml of the CXCR4 antagonist 4F-benzoyl-TE14011 ( 24 ) in saline were implanted dorsally s.c. according to the manufacturer’s instructions. As analgesics, Buprenorphine (0.05–0.1 mg/kg, Sigma-Aldrich) was given i.p. before and after surgery, Carprofen (5mg/kg, Pfizer Animal Health) was given i.p. before surgery and Bupivicaine (100μl of 0.25%, Hospira, Inc.) was given topically during surgery. Tissues were analyzed 12 or 24 hours later. For two-photon laser scanning microscopy (TPLSM) of intact GCs from pLNs and MLNs, Cxcl12 -GFP mice were immunized with SRBC s.c. on day 0 and day 5. On day 8, mice were injected with 2 mg rabbit IgG anti-PE (200-4199, Rockland) i.p. and 12 hours later injected with 75 μg PE (P-801, Invitrogen Molecular Probes) s.c. as previously described ( 25 , 26 ). On day 9, mice were transferred 1.5 x 10 8 CFP transgenic B cells purified from donor spleens using anti-CD43 microbeads (Miltenyi Biotec) i.v. as previously described ( 26 ). PLNs and MLNs were mounted and imaged 24 hours later. For TPLSM of PPs, Cxcl12 -GFP mice were crossed to MD4 mice and transferred with 20% CFP transgenic B cells and 80% CD45.1 WT B cells. B cells were purified from donor spleens as above and 1.2 x 10 7 total B cells were injected i.v. into Cxcl12 -GFP MD4 mice. Two weeks later the mice were injected i.v. with 2 x 10 7 purified CD45.1 WT B cells labeled with CellTracker orange 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR, C2927, Invitrogen) as previously described ( 26 ). Experiment was repeated as above with transfer of 5% CFP transgenic B cells for 4 weeks and 10% CFP transgenic B cells for 2 weeks with similar results. PPs were mounted and imaged 24 hours later.

Confocal Microscopy

Confocal microscopy was preformed as described previously with some modifications ( 15 ). Tissues were fixed in 4% PFA in PBS for 2 hours at 4°C, washed 3 times for 10 min in PBS, then moved to 30% sucrose in PBS overnight. Tissues were flash frozen in TAK tissue-mounting media the following day, and 30μM sections were cut and then dried for 1 hour prior to staining. Sections were rehydrated in PBS with 1% BSA for 10 min and then blocked for 1 hour at room temperature, stained in primary antibody overnight at 4°C and stained for subsequent steps for 2 hours at room temperature all in PBS with 2% mouse serum, 0.1% BSA, 0.3% Triton X-100 and 0.1% NaN3. For gp38 staining, LNs were fixed in 4% PFA in PBS overnight at 4°C, washed 3 times in PBS, then moved to 20% sucrose in PBS overnight. Sections were processed as above except for rinse in PBS and peroxidase quench in PBS with 0.045% H 2 O 2 for 15 min prior to blocking for 30 min. Sections were then stained with primary antibody for 1 hour at room temperature and then with streptavidin–horseradish peroxidase (Jackson Immunoresearch) for 30 min followed by treatment with the TSA Bioin System tyramide staining kit (Perkin Elmer) according to the manufacturer’s instructions. Sections were then stained with remaining secondary antibodies for 1 hour at room temperature. Slides were mounted with Fluoromount-G (Southern Biotech), and images were taken with a Leica SP5 inverted microscope with 40x and 63x oil immersion objectives. Images were analyzed and processed with the Imaris software and the statistics reported are average values with variability represented as standard error of the mean (SEM). Antibodies, Immunofluorescence and Flow Cytometry For immunofluorescence, sections were stained with primary antibodies: Rabbit anti-GFP (A11122, Life Technologies), biotin-conjugated anti-CD35 (8C12, BD Pharmingen), goat anti-mouse IgD (goat polyclonal GAM/IGD(FC)/7S, Cedarlane Labs), APC-conjugated anti-TCRβ (457-597, eBioscience), Alexa647-conjugated anti-Bcl6 (K112-91, BD Pharmingen), biotin-conjugated anti-gp38 (8.1.1, Biolegend), APC-conjugated anti-CD21/35 (7E9, Biolegend), rat anti-mouse CD16/32 (FcγRII/III; UCSF Hybridoma Core), biotin-conjugated anti-FDC-M2 (RmC16D2, Cedarlane Labs), rat anti-mouse FDC-M1 (551320, BD Pharmigen), rabbit anti-PDGFRβ (28E1, Cell Signaling, gift from J. Rock), rabbit anti-laminin (L9393, Sigma), rat anti-VCAM1 (553330, BD Pharmingen), rabbit anti-collagen IV (ab19808, AbCam) and Alexa488 conjugated anti-GFP (A21311, Life Technologies, gift from A. Gerard). Sections were then stained with the following secondary antibodies: Alexa488-conjugated donkey anti-rabbit (A-21206, Life Technologies), Alexa555-conjugated streptavidin (S-21381, Life Technologies), AMCA-conjugated donkey anti-goat (705-156-147, Jackson Immunoresearch), biotin-conjugated donkey anti-rat (712-065-153, Jackson Immunoresearch) and Alexa647-conjugated donkey anti-rabbit (711-606-152, Jackson Immunoresearch). For flow cytometry, single cell suspensions were generated and stained as previously described ( 26 ). The following antibodies were used for cell staining: APC-Cy7-conjugated CD45R/B220 (RA3-6B2, Biolegend), PerCp-Cy5.5-conjugated anti-IgD (11–26c.2a, Biolegend), Alexa647-conjugated anti-T- and B-Cell Activation Antigen (GL7, Biolegend) and PE-Cy7-conjugated Fas (Jo2, BD Biosciences/Fisher). Samples were acquired and analyzed with a BD LSR II and Flowjo (Treestar).

Two-photon laser-scanning microscopy

Explant pLNs, MLNs and PPs were prepared for TPLSM as previously described for explant pLN ( 26 ) except that PP were mounted with the serosal side face-up. PPs were stabilized in a customized plastic coverslip window with Vetbond tissue glue (3M) to counter the peristaltic motion of the small intestine. The temperature at the PP during and at the end of several imaging sessions was measured using a dual-temperature controller (TC-344B, Warner Instruments) equipped with a CC-28 cable containing a bead terminator and was found to remain between 36–37 °C. Images were acquired with ZEN2012-Black Edition (Carl Zeiss) using a 7MP two-photon microscope (Carl Zeiss) equipped with a Chameleon laser (Coherent). For video acquisition from MLNs and pLNs, a series of planes of 0.5μm (MLN) or 1μm (pLN and primary follicle pLN) Z-spacing spanning a depth of 190–260μm were collected. Each XY plane spans 283.40μm × 283.40μm at a resolution of 0.55μm per pixel (MLN, primary pLN) or 327.00μm × 327.00μm at a resolution of 0.64μm per pixel (pLN). Some images have been cropped in XY plane for optimal visualization. Excitation wavelength was 920nm. For video acquisition from PPs, a series of planes of 3μm Z-spacing spanning a depth of 50–100μm were collected every 15–30s. Each XY plane spans 425.10μm × 425.10μm at a resolution of 0.83μm per pixel or 212.55μm × 212.55μm at a resolution of 0.42μm per pixel. Excitation wavelength was 870nm. For all TPLSM imaging, emission filters were