Omentum and milky spots

2. Omentum and milky spots • the omentum is formed by a double layer of • mesothelial cells • connect the stomach, pancreas, spleen and • colon • has immunological and wound-healing properties • embedded within the omentum are structures • which are clusters of leukocytes, called milky • spots

3. Omentum and milky spots • milky spots are mainly composed of macrophages and • B1 cells • B1cells forma unique subset of B cells • can be distinguished from conventional B (B2) cells by • expression of distinct cell-surface markers and antigen • receptors that can bind common bacterial epitopes • have a recognized potential to produce natural • antibodies that provide a first protection to bacterial • infections • B1 cells are localized in distinct locations, such as the • peritoneal cavitiy and the spleen • MS described to lack dendritic cells as well as follicular • dendritic cells

4. Are milky spots secondary lymphoid organs?

5. Mouse system • want to address the immunological potential of milky spots in the • absence of lymph nodes, spleen, and Peyer’s patches • used splenectomized lymphotoxin-alpha (LTa)-deficient mice (Lta-/-), • which as a result of their deficiency already lacked lymph nodes • and Peyer’s patches • animals, devoid of secondary lymphoid organs, were reconstituted • with wild-type bone marrow (SLP mice) • compared to irradiated C57BL/6 mice that were similarly • reconstituted with wild-type bone marrow

6. Antibody responses to peritoneal antigens in the absence of conventional lymphoid organs NP-OVA i.p. TNP-KLH i.p. NP-OVA i.p. • WT and SLP mice produce similar titers of NP-specific IgM, IgG1, IgG2a+b and IgG3 • after immunization with TNP-KLH SLP mice showed slower generation of IgG titer • concentration to see how much secific IgG was produced • conventional lymphoid organs are not needed for B cell response

7. The milky spots of the omentum collect peritoneal cells and antigens HEV + B cell • activation of peritoneal cells promotes their migration to the omentum • at least some particulates can be passively collected by the omentum in addition • to being captured by phagocytic cells

8. The milky spots of the omentum collect peritoneal cells and antigens • peritoneal cells use PTX-sensitive mechanisms to actively migrate to the omentum • can also accumulate in the omentum by other mechanisms • the segregation of B and T cells and the formation of follicular structures in the MSs • are controlled by PTX-sensitive mechanisms

9. Antigen-specific B cell responses occur in the omentum NP-OVA i.p. SRBC i.p boost d14 GC B cells GC B cells Isotype-switched plasmacells SRBC specific IgG secreting cells specific non-specific d8 d5

10. Antigen-specific B cell responses occur in the omentum SRBC i.p d14 NP-OVA i.p d14 GC B cells • MSs support local germinal center B cell responses to peritoneal antigens

11. Antigen-specific T cell responses in the omentum T cell activation homing receptor OTII CD4 i.p. 4 hr later immunized • T cell responses can also occur • in the MSs of the omentum, • even in mice that lack spleen, • LNs, and Peyer’s patches

12. Antigen-specific T cell responses in the omentum influenza i.n. 21d p.i. 4-get mice helminth larvae oral 28 d p.i. • antigen-experienced CD4+ and CD8+ T cells recirculate through the • peritoneal cavity and omentum • even when these cells were primed outside of the peritoneal cavity

13. Milky spot development requires LTα and CXCL13, but not LTi cells inactive genes for CCL19, CCL21 • MSs were much smaller or even absent in Lta-/- and Cxcl13-/- mice • MSs were much smaller or even absent in Lta-/- and Cxcl13-/- mice • defects in theMSs of Lta-/- mice seem not be due to an absolute blockade • in development

14. Milky spot development requires LTα and CXCL13, but not LTi cells lymphoid tissue inducer cells • even though CXCL13 is very important for the development of the MSs, • its expression is not controlled by Lta • Lti cells are not needed

15. CXCL13 is expressed surrounding the B cell areas omentum spleen • CXCL13 is expressed around the B cell area • absence of conventional FDC networks

16. CXCL13 is expressed surrounding the B cell areas • network of ERTR7+ stromal cells throughout the MSs • the cellular architecture and pattern of chemokine expression in the MSs • are different than in conventional lymphoid organs

17. Somatic hypermutation and affinity selection occurs in the absence of conventional secondary lymphoid organs NP-OVA i.p. boost d14 • W to L position 33 • higher affinity • YYYG motif at V-D • junction • NP-binding sequence • somatic hypermutation is still evident in SLP mice • but the process of clonal selection is unusual • the MSs of the omentum support some aspects of T cell dependent • B cell responses

18. Summary • the omentum functions much more broadly as a secondary • lymphoid organ • it is structurally, developmentally, and functionally unique • intersection of recirculating lymphocytes and peritoneal drainage makes the • MSs ideal sites for the initiation of local immune responses • the structure of the MSs seems inside-out relative to that of conventional • lymphoid organs or even ectopic lymphoid follicles • the MSs of the omentum are unique secondary lymphoid organs that sample antigens from the peritoneal cavity and promote local, albeit • unusual, immune responses