Kevin J. Comerford Assistant Professor/Digital Initiatives Librarian Jonathan Wheeler
Atypical chemokine receptor 4, ACKR4, in B cell activation€¦ · Biomed Rep 1(2): 185-192...
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Primary as wellas secondary immunization of ACKR4 ko mice with protein antigens initially results in normal plasma cell and GC B cell numbers. Also, there are no differences in antibody titres measured by ELISA. However, ACKR4 ko mice show GCs with abnormal shape, with more naïve B cells entering the GC periphery. Bajoghli, B. (2013). Eur J Immunol 43(7): 1686-1692 Chew, AL. (2013). Biomed Rep 1(2): 185-192 Comerford, I., et al (2006). Eur J Immunol 36(7): 1904-1916 Ulvmar, M. H., et al (2011). Exp Cell Res 317(5): 556-568 References The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement n°315902. Acknowledgements Laura Garcia Ibanez 0121 414 6970 [email protected] Contact Atypical chemokine receptors have been recently described as regulators of chemokine signalling. ACKR4, or previously known CCRL1, is one of the four components of this family (Ulvmar, 2011). ACKR4 is able to internalise its ligands (CCL19, CCL21 and CCL25. CXCL13 has only been confirmed in human ACKR4), regulating their availability outside the cells (Comeford, 2006). ACKR4 is expressed in heart, lung, small intestine, brain, testes and lymph nodes. In the lymph nodes ACKR4 is expressed in the subcapsular sinus, creating a gradient of CCL19, influencing the movement of DCs towards the T zone (Ulvmar, 2014). ACKR4 deficient mice present higher incidence of autoimmune diseases such a autoimmune encephalomyelitis (Comerford, 2010). The expression of ACKR4 has been described as a negative marker for metastases in multiple cancer models (breast, colorectal and squamous cell carcinoma). Atypical chemokine receptor 4, ACKR4, in B cell activation L. Garcia Ibanez (1) , S.L. Cook (1) , J.C. Yam-Puc (2) , Y. Zhang (1) , G. Brown (1) , A. Rot (3) , K.M. Toellner (1) (1) School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, UK (2) Centre for Advanced Research, The National Polytechnic Institute, Cinvestav-IPN, Mexico City, Mexico (3) Centre for Immunology and Infection, University of York, UK Determine the role of the atypical chemokine receptor ACKR4 in the secondary lymphoid organs after TD immunisation and therein in the GC response. Aim Introduction • The deficiency of ACKR4 is not influencing the B cell response (Germinal centre B cell numbers, plasma cell numbers and antibody titres) at the peak of the Germinal Centre. • However, the expression of ACKR4 in non-B cells influences GC shape, diminishing the area free of naïve B cells within the Germinal centre area and the distribution of antigen-specific B cells.. • Although this shape modification is not affecting GC output in the peak of the GC response, it could be having an effect at later stages such as maintenance and disappearance of the GC. Conclusions ACKR4 is expressed in GC B cells at mRNA and protein level, both in B cells and in FDCs. In non-immunised mice, ACKR4 is not expressed in any haematopoietic cells, but is expressed in the subcapsular sinus of the lymph nodes (Ulvmar, 2014) and in the splenic red pulp stroma surrounding the follicles. Expression ACKR4 het and ACKR4 ko response to TD antigen When ACKR 4 deficient 4-hydroxynitrophenyl-specific (NP) eYFP + B cells (QM) are transferred to wild type hosts and immunized with NP-Ficoll, GC shape is restored. Transfer of ACKR4 ko QM B cells to WT environment When ACKR 4 sufficient 4-hydroxynitrophenyl-specific (NP) eYFP + B cells (QM) are transferred into ACKR 4 deficient hosts, GC shape is disturbed again. Transfer of ACKR4 wt QM B cells to ACKR4 ko environment 10 -5 10 -4 10 -3 10 -2 ACKR4 Day 8 after immunisation with NP-CGG Relative expression to 2-Microglobulin < ●: Naïve B cells (B220 + ) : GC B cells (B220 + NP + CD38 - Fas + ) : Memory B cells (B220 + NP + CD38 + ) NP /IgD WT KO TZ F RP F RP a) b) d) Figure 2. a) Splenic flow cytometry data of plasma cells (B220 + NP + CD138 + ) (left) and GC B cells (B220 + NP + CD38 - Fas + ) (right) from ACKR4 het and ACKR4 ko mice at day 8 after immunisation with NP-CGG. b) Splenic flow cytometry data of plasma cells (left) and GC B cells (right) from ACKR4 het and ACKR4 ko mice at day 5 after boost immunisation with NP-CGG. c) Relative antibody titres from ACKR4- het and ACKR4- ko mice immunised like in a) (upper) and like in b) (lower). d) Representative immunohistochemistry staining images of spleens from ACKR4 het mice (left) and ACKR4 ko mice (right) immunised like a). Key. F: B follicle; TZ: T zone; RP: red pulp ●: ACKR4 het ○: ACKR4 ko c) eYFP/PNA/IgD ●: ACKR4 wt QM B cells ○: ACKR4 ko QM B cells Figure 3. a) Representative immunofluorescence image of a WT spleen transferred with ACKR4 ko QM B cells 4 days after immunisation with NP-Ficoll. b) Ratio of PNA + area / IgD - area a) b) eYFP/PNA/IgD a) b) ●: ACKR4 het recipient ○: ACKR4 ko recipient Figure 4. a) Representative immunofluorescence image of a ACKR4 deficient spleen transferred with ACKR4 sufficient QM B cells 4 days after immunisation with NP-Ficoll. b) Ratio of PNA + area / IgD - area. ACKR4/CXCR4/IgD ACKR4/FDCM2 GC TZ F RP a) b) c) Figure 1. a) qPCR data of ACKR4 expression in different B cell subsets obtained by sorting from lymph nodes of WT mice transferred with Cγ1-Cre x QM ROSAeYFP B cells 8 days after immunisation with NP-CGG in the footpads. b) and c) Representative immunofluorescence staining images of a WT spleen 8 days after immunisation with SRBC. b) ACKR4 expression in GC B cells. c) ACKR4 expression in FDCs. Key. F: B follicle; TZ: T zone; RP: red pulp. GC: germinal centre ●: ACKR4 het ○: ACKR4 ko 100 1000 10000 100000 Plasma cells Day 8 after immunisation with NP-CGG Absolute cell count 10000 100000 1000000 Germinal Centre cells Day 8 after immunisation with NP-CGG Absolute cell count 100000 1000000 Plasma cells Primed with CGG Day 5 after immunisation with NP-CGG Absolute cell count 100 1000 10000 Germinal Centre cells Primed with CGG Day 5 after immunisation with NP-CGG Absolute cell count 10 100 1000 10000 ELISAS Day 8 after immunisation with NP-CGG Antibody titre 10 100 1000 10000 100000 1000000 ELISAS Primed with CGG Day 5 after immunisation with NP-CGG Antibody titre TZ 0.0 0.5 1.0 1.5 2.0 2.5 GC area Day 4 after immunisation with NP-Ficoll PNA + area / IgD - area **** 0.0 0.5 1.0 1.5 GC area Day 4 after immunisation with NP-Ficoll PNA + area / IgD - area (au)
Transcript of Atypical chemokine receptor 4, ACKR4, in B cell activation€¦ · Biomed Rep 1(2): 185-192...
Primary as well as secondary immunization of ACKR4 ko mice with protein antigens initially results in normal plasma cell and GC B cell numbers. Also, there are no differences in antibody titresmeasured by ELISA. However, ACKR4ko mice show GCs with abnormal shape, with more naïve B cells entering the GC periphery.
Bajoghli, B. (2013). Eur J Immunol 43(7): 1686-1692Chew, AL. (2013). Biomed Rep 1(2): 185-192
Comerford, I., et al (2006). Eur J Immunol 36(7): 1904-1916Ulvmar, M. H., et al (2011). Exp Cell Res 317(5): 556-568
ReferencesThe research leading to these results has received funding from the PeopleProgramme (Marie Curie Actions) of the European Union’s Seventh FrameworkProgramme FP7/2007-2013/ under REA grant agreement n°315902.
AcknowledgementsLaura Garcia Ibanez0121 414 [email protected]
Contact
Atypical chemokine receptors have been recently described as regulators of chemokinesignalling. ACKR4, or previously known CCRL1, is one of the four components of this family(Ulvmar, 2011).ACKR4 is able to internalise its ligands (CCL19, CCL21 and CCL25. CXCL13 has only beenconfirmed in human ACKR4), regulating their availability outside the cells (Comeford,2006).ACKR4 is expressed in heart, lung, small intestine, brain, testes and lymph nodes. In thelymph nodes ACKR4 is expressed in the subcapsular sinus, creating a gradient of CCL19,influencing the movement of DCs towards the T zone (Ulvmar, 2014).ACKR4 deficient mice present higher incidence of autoimmune diseases such aautoimmune encephalomyelitis (Comerford, 2010). The expression of ACKR4 has beendescribed as a negative marker for metastases in multiple cancer models (breast, colorectaland squamous cell carcinoma).
Atypical chemokine receptor 4, ACKR4, in B cell activationL. Garcia Ibanez (1), S.L. Cook (1), J.C. Yam-Puc (2), Y. Zhang (1), G. Brown (1), A. Rot (3), K.M. Toellner (1)
(1) School of Immunity and Infection, College of Medical and Dental Sciences, University of Birmingham, UK(2) Centre for Advanced Research, The National Polytechnic Institute, Cinvestav-IPN, Mexico City, Mexico(3) Centre for Immunology and Infection, University of York, UK
Determine the role of the atypical chemokine receptor ACKR4 in the secondary lymphoidorgans after TD immunisation and therein in the GC response.
AimIntroduction
• The deficiency of ACKR4 is not influencing the B cell response (Germinal centre B cellnumbers, plasma cell numbers and antibody titres) at the peak of the Germinal Centre.
• However, the expression of ACKR4 in non-B cells influences GC shape, diminishing thearea free of naïve B cells within the Germinal centre area and the distribution ofantigen-specific B cells..
• Although this shape modification is not affecting GC output in the peak of the GCresponse, it could be having an effect at later stages such as maintenance anddisappearance of the GC.
Conclusions
ACKR4 is expressed in GC B cells at mRNA and protein level, both in B cells and in FDCs.In non-immunised mice, ACKR4 is not expressed in any haematopoietic cells, but isexpressed in the subcapsular sinus of the lymph nodes (Ulvmar, 2014) and in the splenicred pulp stroma surrounding the follicles.
Expression
ACKR4het and ACKR4ko response to TD antigen
When ACKR4 deficient 4-hydroxynitrophenyl-specific (NP) eYFP+ B cells (QM) aretransferred to wild type hosts and immunized with NP-Ficoll, GC shape is restored.
Transfer of ACKR4ko QM B cells to WT environment
When ACKR4 sufficient 4-hydroxynitrophenyl-specific (NP) eYFP+ B cells (QM) aretransferred into ACKR4 deficient hosts, GC shape is disturbed again.
Transfer of ACKR4wt QM B cells to ACKR4ko environment
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Figure 4. a) Representative immunofluorescence image of a ACKR4 deficient spleen transferred with ACKR4 sufficient QM B cells 4 days after immunisation with NP-Ficoll. b) Ratio of PNA+ area / IgD- area.
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Figure 1. a) qPCR data of ACKR4 expression in different B cell subsets obtained by sorting from lymph nodes ofWT mice transferred with Cγ1-Cre x QM ROSAeYFP B cells 8 days after immunisation with NP-CGG in thefootpads. b) and c) Representative immunofluorescence staining images of a WT spleen 8 days afterimmunisation with SRBC. b) ACKR4 expression in GC B cells. c) ACKR4 expression in FDCs.Key. F: B follicle; TZ: T zone; RP: red pulp. GC: germinal centre
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