Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position Karin Reif, Eric H. Ekland, Lars Ohl, Hideki Nakano, Martin Lipp, Reinhold Forster & Jason G. Cyster Nature 416: Presented by: Michelle Liu, Michael Marit, Eddie Poon

Chemokine Background Diagram of generalized chemokine receptor: Chemokine Receptor Chemokine G Protein Chemotactic Signals

CXCR5 Background - expressed on B cells at a constant level throughout activation - responds to CXCL13, a ligand expressed by the B cell follicle - ligation of CXCL13 by CXCR5 allows for B cell entry into follicles Key points concerning CXCR5:

CCR7 Background Key points concerning CCR7: - expression guides lymphocytes to T cell zones in secondary lymphoid tissues (Janeway, 417) - has been identified as a major homing receptor for B cells, T cells, and dendritic cells (Forster et al., 1999) - expression further localizes B cells to the T zone which promotes T cell – B cell interaction (Janeway, 349) - ligates CCL19 and CCL21

CCR7 Background - First introduced by Dr. Kelvin on February 1st - Good background reading: Forster et al. Cell 99, 23 – 33. (1999). – Introduced by Dr. Kelvin Campbell et al. Immunological Reviews, 195, 58 – 71. (2003). CCR7 Coordinates the Primary Immune Response by Establishing Functional Microenvironments in Secondary Lymphoid Organs Chemokines in the Systemic Organization of Immunity

Campbell et al.: CCR7 plays a role in lymphocyte trafficking to secondary lymphoid organs CCR7 Background Regular B and T cell migratory patterns Upregulation by B cells targets them to B/T boundary Lymph Nodes Spleen PPs Figure obtained from Campbell et al., 2003, modified.

Transgenic Mice - Experiments used cells from mice carrying immunoglobulin transgenes specific for hen-egg lysozyme (abbreviated Ig HEL ) - Introduction of transgene carrying pre-rearranged heavy and light chains specific for the hen-egg lysozyme - Isolation of transgenic B cells carrying the pre-arranged immunoglobulin receptor, introduction to congenic mouse of a different immunoglobulin allotype

Figure 1a) Question: After antigen stimulation, what is the endogenous rate of B cell migration to the B-zone/T-zone (B/T) boundary in the spleen? Experiment: Introduce transgenic B cells, take sections of spleen tissue just after antigen stimulation, 3 hours after, 6 hours after.

Figure 1a) Experiment: Sections taken of spleen tissue before antigen stimulation, 3 hours after, 6 hours after. Conclusion: Migration of antigen-engaged B cells to the B/T boundary in the spleen is complete in 6 hours post-injection.

Figure 1b) Question: How does the CCR7 expression level change from B cell activation? Experiment: Flow cytometry - in vivo injection of HEL antigen or in vitro anti-IgM antibody incubation, 6 hour lapse - CCL19-Fc (CCR7 ligand) used as reporter to test CCR7 expression levels

Figure 1b) Experiment: Flow cytometry Conclusion: Six hours after antigen exposure, B cells showed a two- threefold CCR7-expression increase. Therefore during activation, CCR7 upregulation occurs, and CXCR5 expression remains the same.

Figure 1c) Question: How is the chemotactic responsiveness affected by an increase of CCR7 expression? Problem: HEL antigen binds strongly to the transwell filter, therefore in vitro activation of B cells using the HEL antigen cannot be isolated for in vivo transfer and study. Solution: Use IgM-based activation of B cells and transfer to a congenic mouse to visualize migration patterns.

Figure 1c) Question: How is the chemotactic responsiveness affected by an increase of CCR7 expression? Experiment: Isolation of IgM a B cells, incubation with anti-IgM antibody (activates B cells), transfer to a congenic IgM b mouse, and observe localization via allotype-specific antibody

Figure 1c) Experiment: Isolation of IgM a B cells, incubation with anti-IgM antibody (activates B cells), transfer to a congenic IgM b mouse, and observe localization via allotype-specific antibody. Conclusion: The anti-IgM treatment activated B cells, and caused localization to the B/T boundary in the spleen.

Figure 1d) Question: What is the migratory behaviour of the anti-IgM- stimulated spleen cells ex vivo? Experiment: Chemotaxis assay - determines the chemotactic response to a specific molecule - a gradient of the molecule is created and the migratory behaviour of a cell is analyzed

Figure 1d) Conclusion: Chemotactic migration in response to CCL19 and CCL21 (CCR7 ligands) is greatly increased in activated B cells as compared to endogenous and control. Experiment: Chemotaxis assay

Experiment: - take plt/plt mice that are deficient in the CCL19 and the lymphoid tissue form for CCL21 - transfer Ig HEL -transgenic B cells to mice - observe migration patterns of B cells Question: Does antigen-mediated relocalization of B cells to the T zone require B cell expression of CCR7 and response to chemokines CCL19 and CCL21 in vivo? Figure 2a)

In the absence of antigen, B cells localized in follicles stimulated B cells localized at the B/T boundary stimulated B cells remain in follicles - HEL + HEL - due to altered T zone? - B cell, HEL binding, CCR7-/- stimulated B cells fail to localize at the B/T boundary – B cells often moved in the opposite direction Conclusion: CCR7 and its ligands CCL19 and CCL21 allow T/B boundary localization Figure 2a) CCL19 and CCL21 deficient

The ROLE of CXCR5 and its ligand CXCL13 in B cell relocalization to T zone in vivo Experiment: transfer CXCR5 -/- Ig HEL -transgenic B cells into mice and stimulate with HEL antigen observe migration patterns of B cells Figure 2b)

- HEL + HEL CXCR5 -/- B cells were found localized at red pulp and entry points to T Cell areas, very little in B Cell follicles. Stimulated B Cells with HEL - increased cell accumulation at the B Cell / T Cell boundary, unevenly distributed often as clusters near bridging zones. Conclusion: CXCR5/CXCL13 interaction not required for B cell migration to T zones but influences the localization helping distribution along the B/T boundary Figure 2b)

Model: BCR signalling induces CCR7, the determinant for B Cell relocalization to T zone Experiment: Ig HEL -transgenic B cells incubated with mitogenic doses of anti- CD40 Ab  ligation drives cell cycle/ proliferation... and production of new proteins retroviral-mediated gene transfer vectors in B cells selectively alter chemokine receptor levels in B cells adoptive transfer in mice. MSCV B B Figure 3ab)

Flag tagged CCR7 with marker protein – cytoplasmic domain truncated human CD4 B cells activated by mitogenic doses of anti-CD40 and infected with the vector do not undergo major changes in expression of: Flow Cytometry Figure 3ab) CCR7 or CXCR5

Ex vivo flow cytometry of B cells from Ig HEL B cells transduced with vectors CCR7 vector Increased CCL19-Fc binding Vectors express hCD4 Flag epitope ‘M2’ – indicator shows CCR7-flag tagged expression from the vector Figure 3c) Conclusion: Vector works in CCR7 expression, and also allows transduced cells to bind with CCR7 binding targets

Ex vivo chemotaxis Analysis of Ig HEL -transduced B cells Higher CCR7 expression resulted in increases CCL21 sensitivity Higher CCR7 expression resulted in increases CCL19 sensitivity. Figure 3d) Greater migratory outcome than BCR stimulated cells confirms higher CCR7 expression in transduced cells

hCD4 used as a surrogate marker for B cells that overexpress CCR7 in situ B cells localize in follicles (similar to nontransduced cells) CCR7-transduced B cells distribute in the T zone CCR7-transduced B cells distribute at the B/T boundary and in the T zone CCR7-transduced B cells remain in follicles Figure 3e) Conclusion: Implies that activated B cells use CCR7 and CCL19/CCL21 to move towards T zone, and CXCR5/CXCL13 to accumulate at B/T boundaries

Question: Does antigen-mediated relocalization of B cells to the T zone depend on a balance of chemokine responsiveness? Experiment: - Test: Is overexpression of CXCR5 by retroviral transduction sufficient to overcome effect of antigen engagement on B cell distribution? Figure 4) - activate Ig HEL -transgenic B cells by incubation with mitogenic doses of anti- CD40 Ab - use retroviral-mediated gene transfer to selectively alter CXCR5 receptor levels in B cells - adoptive transfer in mice

Ex vivo chemotaxis assay - B cells overexpressing CXCR5 show increased responsiveness to CXCL13 Increased expression of CXCR5 following retroviral transduction with a CXCR5 vector gene compared to the empty vector (control) Figure 4ab)

Ag-stimulation and BCR activation fail to promote follicular exclusion B cells are found in follicles (where CXCL13 is expressed) Ag-stimulation and BCR activation promotes localization at the B/T boundary Prior to Antigen stimulation, B cells localize in B cell follicles (in control and cells overexpressing CXCR5) Conclusion: Overexpression of CXCR5 is sufficient to override antigen-induced B-cell movement to the T-cell zone. Figure 4c)

This paper establishes that: The positioning of B cells in lymphoid tissues in vivo is determined by relative responsiveness of B cells to chemokines made in separate but adjacent zones (T zone vs. B zone)

Summary CCL19 and CCL21 deficient mice  B cells do not relocalize to the B/T boundary CCR7-/- mice  B cells do not relocalize to the B/T boundary CXCR5-/- mice  when unstimulated with Ag, B cells do not enter follicles  when stimulated with Ag, increased but uneven B cell relocalization at the B/T boundary Increased CCR7 expression  increased B cell relocalization at (due to retroviral transduction) the B/T boundary Increased CCR7 expression  increased B cell relocalization & CXCR5-/- in the T zone Increased CXCR5 expression  B cells remain in follicles (due to retroviral transduction)

B cells enter spleen and LNs through blood vessels outside B cell zones Naive B cells express high levels of CXCR5 and low levels of CCR7  B cells migrate into follicles due to favoured responsiveness to CXCL13 Ag-stimulated B cells (following BCR engagement), B cells upregulate expression of surface CCR7  B cells redistribute to the T zone (for help by T H 2 cells) due to favoured responsiveness to T-zone chemokines (CCL19 and CCL21)  B cells integrate signals from two chemokine receptors (CCR7 and CXCR5) simultaneously which results in localization at the B/T boundary

Vessel Antigen B zone (follicle) T zone B cell CCL19 & CCL21CXCL13 T zone stromal cell B zone stromal cell CXCR5 T H 2 cell CCR7

Ag-stimulated B cells respond less strongly than T cells and DC to CCL19 and CCL21  B cells are excluded from the central T zone T H 2 cells express less CCR7 than other T cell subtypes  T H 2 cells are excluded from the central T zone and are more accessible to help B cells Future direction:  contribution of additional factors (ie. adhesion molecules)  study positioning of other cell types due to balanced responsiveness to chemoattractants from produced in adjacent zones Autoantigen engaging B cells also localize at the B/T boundary

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