A–F. Omenta were obtained from naïve C57BL/6 mice and cryosections were probed with the indicated antibodies. G–J. Spleens were obtained from naïve C57BL/6 mice and cryosections were probed with the indicated antibodies. Cryosections in panels B–E were stained at the probed at the same time, under the same conditions as cryosections in panels G–J. K–L. Omenta were obtained from naïve C57BL/6 mice and whole mounts were probed with the indicated antibodies. Images were generated using a combination of fluorescence (optical sections using the Zeiss apotome) and DIC. Control primary and secondary antibodies were negative. Images are representative of 3–5 mice.

A–B. WT and SLP mice were i.p. immunized with NP-OVA. The serum titers of NP-specific IgM and total IgG (A) as well as NP-specific IgG1, IgG2a, IgG2b and IgG3 (B) were determined by ELISA. This experiment is representative of two experiments with 5 mice in each group. C. WT and SLP mice were immunized with TNP-KLH. The serum titers of TNP-specific IgG were determined by ELISA and the concentration of TNP-specific IgG was determined by comparison to a monoclonal anti-TNP standard. This experiment was performed twice with similar results. D. C57BL/6 and splenectomized Lta−/− mice were immunized with NP-OVA. The serum titers of NP-specific IgG were determined by ELISA

A. Omenta were obtained from naïve C57BL/6, Lta^−/−, Cxcl13^−/− or plt/plt mice as indicated and whole mounts were probed with antibodies to B220 and CD11b. B. Paraffin sections of omenta were stained with H&E. C. Paraffin sections of omenta were probed with antibodies to B220 and PNAd. D. RNA was extracted from the omenta of naïve C57BL/6, Lta^−/−, Cxcl13^−/− or plt/plt mice as indicated and assayed for the expression of CXCL12, CXCL13, CCL19, CCL21, LTβ and TNFα by quantitative PCR. The expression of each mRNA was normalized first to GAPDH and then normalized to the expression in WT animals, which was set at 1. E. Omenta were obtained from naïve Rorc^−/− or Id2^−/− mice as indicated and paraffin sections were stained with H&E (E) or probed with antibodies to B220 and CD11b (F) or B220 and CD31. Images are representative of 3–5 mice per group.

A. WT and SLP mice were i.p. immunized with NP-OVA and boosted on day 14. RNA was extracted from omenta on days 7, 14 and 19 after immunization. The V regions of B cells with V186.2 heavy chains that had switched to IgG1 were amplified by PCR, subcloned and sequenced. The pie charts indicate the relative proportion of sequences that were unmutated (0) or contained the indicated number of mutations. B. The titers of NP-specific total IgG were determined using ELISA plates coated with either NP₁₆-BSA and with NP₂-BSA. The relative affinity of the NP-specific IgG is expressed as a ratio of binding to the NP₂-BSA versus NP₁₆-BSA. There were 5 mice in each group and this experiment is representative of 3 independent experiments.

A–B. WT and SLP mice were i.p. immunized with NP-OVA and omental cells were analyzed by flow cytometry on day 8 for NP-specific FAS⁺PNA-binding germinal center B cells (A) and NP-non-specific FAS⁺PNA-binding germinal center B cells (B). Cells in A–B were gated on CD19 expression and the ability to bind NP-APC (A) or the failure to bind NP-APC (B). The numbers in each plot refer to the percentage ± standard deviation of NP-specific B cells in the gates indicated by ovals or boxes. C–D. WT and SLP mice were i.p. immunized with SRBC, boosted on day 14 and omental cells were analyzed 5 days later by flow cytometry for FAS⁺PNA-binding germinal center B cells (C) and IgG1⁺CD138⁺ plasma cells (D). Cells in C–D were gated on CD19 expression. The numbers in each plot refer to the percentage ± standard deviation of CD19⁺ B cells in the gates indicated by ovals or boxes. E. WT and SLP mice were i.p. immunized with SRBC, boosted on day 14 and omental cells were analyzed by SRBC-specific ELISPOT. F. The frequency and total number of SRBC-specific IgG ELISPOTs was determined. There were 4–5 mice in each group. G. C57BL/6 mice were immunized with SRBC and omenta were collected on day 14. Omenta were embedded in pieces of liver to aid in sectioning. Paraffin sections were stained with H&E. H–N. C57BL/6 mice were immunized with NP-OVA and omenta were collected on day14 and frozen in OCT. Cryosections were stained with the indicated antibodies and counterstained with DAPI (blue). Germinal centers are outlined by yellow in panels K–N.

A–C. Purified OTII CD4 T cells were labeled with CFSE and i.p. injected into WT and SLP recipient mice. Recipients were immunized 4 hours later with 100 μg OVA and 10 μg LPS adsorbed to alum or with 10 μg LPS and alum as indicated. Omenta were obtained 36 or 42 hours later and omental cells were analyzed by flow cytometry for CD4, CFSE and CD25 (A) or CD4, CFSE and CD62L (B). The plots shown are gated on CD4⁺ cells. CFSE dilution in the transferred CD4 T cells was measured by flow cytometry (C). There were 4–5 mice per group and omenta were pooled prior to analysis. The data shown is representative of 3 independent experiments. D. WT and SLP mice were immunized with 100 μg OVA and 10μg LPS adsorbed to alum. OVA-specific CD8 T cells were identified by tetramer binding in the omenta of immunized mice. The plots shown are gated on CD8 T cells. E. C57BL/6 mice were intranasally infected with influenza and single cell suspensions from peritoneal cavity and omenta were analyzed by flow cytometry 21 days after infection. Histograms show the frequency of CD8 cells in the live leukocyte population. Dot plots were gated on CD8 cells and the boxes indicate influenza nucleoprotein-specific cells. F. 4-get IL-4 reporter mice were orally infected with H. polygyrus larvae and single cell suspensions from peritoneal cavity and omenta were analyzed by flow cytometry 28 days after infection. Histograms show the frequency of CD4 cells in the live leukocyte population. Dot plots were gated on CD4 cells and the boxes indicate Th2 cells based on GFP expression (B).

A–B. Omenta were obtained from naïve WT and SLP mice and whole mounts were probed with antibodies to B220 and PNAd. Images were obtained as optical sections using the Zeiss Apotome. C–D. C57BL/6 mice were i.p. immunized with ovalbumin coupled to Alexa-594. Omenta were collected 2 or 4 hours later and whole mounts were probed with antibodies to CD11b. E. 1 × 10⁶ CFSE-labeled SRBC were i.p. injected and the omenta were collected 4 hours later. Whole mounts were probed with antibodies to CD11b. The high magnification image is an optical section obtained with the Zeiss Apotome. F. Omental cells from SRBC-injected and control mice were analyzed by flow cytometry. The plots show all events collected. The inset in the top panel shows the profile of CFSE-SRBC prior to injection. The numbers in each plot refer to the percentage ± standard deviation of events in the CFSE-SRBC gate. G. Omental cells from SRBC-injected and control mice were analyzed by flow cytometry. The plots show live leukocytes and gated into 5 populations based on CD11c and CD11b expression. H. Total cells from the omenta of SRBC-injected and control mice were enumerated and the number of cells in each of the gated populations was calculated. I. GFP-expressing EL4 cells were cultured in vitro for 2 hours with or without pertussis toxin and then i.p. injected into C57BL/6 recipients. Omenta were collected 2 or 4 hours later and whole mounts were examined by fluorescence microscopy. J. Whole mounts of omenta from EL4-GFP-injected mice were probed with B220 to visualize MS. Images are optical sections obtained using the Zeiss Apotome. K. The frequency of PTX-treated and untreated EL4-GFP cells in the omenta was determined by flow cytometry. L The number of PTX-treated and untreated EL4-GFP cells in the omenta was calculated. There were 4–5 mice per group. M. B cells were purified from GFP-transgenic mice and mixed 2:1 with purified, bodipy-labeled T cells from C57BL/6 mice. The cell mixture was cultured for 2 hours with or without pertussis toxin and 1 × 10⁷ total cells/mouse were i.p. injected into C57BL/6 recipients. The omenta were collected 2 and 4 hours after injection and whole mounts were analyzed by fluorescence microscopy. N. The frequencies of PTX-treated and untreated B and T cells in the omenta were determined by flow cytometry. O The numbers of PTX-treated and untreated B and T cells in the omenta were calculated. There were 4–5 mice per group.