Regulation of Immune Responses by Proteoglycan/Chemokine interactions: from structural to in vivo approaches – ChemImmun

In this project we used Flow Cytometry and Histology to analyse the structure of the Germinal Center reaction in mutant mice and to identify the cells producing CXCL12 in peripheral lymphoid organs.
Serologic approaches (Enzyme-linked immunosorbent assays – ELISA) were used to quantify the serum antibody response, as well as its affinity, towards model antigens.
Mutated variants of CXCL13 were produced and expressed as recombinant proteins. They were then analysed by nuclear magnetic resonance (NMR) spectroscopy and surface plasmon resonance (SPR) to determine their ability to bind to Heparan Sulfate (HS). The ability of these mutant variants to bind and activate the specific CXCL13 receptor (ie : CXCR5) on the surface of responding cells was determined by standard migration assays using live cells.

Our results reveal the importance of CXCL12 immobilisation in the quality of the humoral immune response. During the GC reaction, immobilised CXCL12 forms a fixed gradient with higher concentration of the chemokine in the Dark Zone. Opposing gradients of CXCL12 and CXCL13 allow B cells to migrate between the Dark Zone and the Light Zone, by alternating expression of its receptor CXCR4. B cells selected in the Light Zone for higher affinity to antigen return to the Dark Zone where they undergo further rounds of proliferation and somatic mutation, before returning to the Light Zone for additional cycles of selection. Disruption of CXCL12 binding to HS prevents the establishment of the fixed gradient needed to direct selected cells back to the Dark Zone, thus impairing the mechanism of step-wise selection for cells carrying increasing affinity to antigen. In consequence, antibody responses are sub-optimal, showing impaired affinity maturation.
This work also shows that CXCL13 binding to extra-cellular matrix components can also be abrogated, with no measurable impairment in CXCR5 binding. Thus, the paradigm we had established for CXCL12 (namely : distinct and non-overlapping domains responsible for binding to the chemokine receptor and the extra-cellular matrix) is also applicable to CXCL13

The achievements of this project provide critical insights into the importance and role of immobilization of CXCL12 on ECM in the immune response and pave the way for a more complete exploration of the share of immobilization vs. diffusion of chemokines in cell trafficking within GCs, with particular significance of areas of different concentrations and isoforms.
In particular, the results open up the possibility of performing structure/fucntion expeirments with CXCL13, similar to those carried out for CXCL12.

PUBLICATIONS

Pegeot P., Sadir R., Eriksson I., Kjellen L., Simorre J.P., Gans P., and lortat-Jacob H. (2015). Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture 13C-glucose metabolic labelling. Glycobiology 25, 151-156

Monneau Y., Arenzana-Seisdedos F. and Lortat-Jacob H. (2016). The sweet spot: how GAGs help chemokines guide migrating cells. Journal of leukocyte Biology 99, 935-953

Monneau Y.R., Luo L., Sankaranarayanan N.V., Nagarajan B., Vivès R.R., Baleux F., Desai U.R., Arenzana-Seisdedos F. and Lortat-Jacob H. (2017). Solution structure of CXCL13 and heparan sulphate binding show that GAG binding site and biological activity rely on distinct domains. Open Biology 7, 170133

Barinov, A., L. Luo, P. Gasse, V. Meas-Yedid, E. Donnadieu, F. Arenzana-Seisdedos, and P. Vieira. (2017). Essential role of immobilized chemokine CXCL12 in the regulation of the humoral immune response. Proc Natl Acad Sci U S A. 114:2319-2324.

Huang HY, Rivas-Caicedo A, Renevey F, Cannelle H, Peranzoni E, Scarpellino L, Hardie DL, Pommier A, Schaeuble K, Favre S, Vogt TK, Arenzana-Seisdedos F, Schneider P, Buckley CD, Donnadieu E*, Luther SA*. (2018) Identification of a new subset of lymph node stromal cells involved in regulating plasma cell homeostasis. Proc Natl Acad Sci U S A 2018 115(29):E6826-E6835. * contributed equally to this work

We developed during the last years an ambitious research project aimed at exploring the biological functions of chemokine immobilization in vivo. This phenomenon is based on the chemokine ability to interact with the glycanic moiety of proteoglycans, and in particular with Heparan Sulfates (HS). Immobilization on HS restrains chemokine diffusion and spatial distribution, thus facilitating the formation of local gradients of chemokine that may regulate the synchronous coordination of cell motility and integrin-dependent cell adhesion. The research accomplished focused on the unique and essential chemokine CXCL12 and has been conducted with the support of the ANR to the projects CHEMOGLYCAN (2005) and CHEMREPAIR (2010) which enabled successively the elucidation of the biochemical and structural basis of chemokine/HS interactions and provided the knowledge required for the generation of the first animal model, investigating in vivo the role of this interaction. The engineered mouse carries a Cxcl12 gene (Cxcl12gagtm) encoding mutations that preclude interactions with HS structures while they do not affect CXCR4-dependent cell-signaling. Cxcl12gagtm/gagtm mice develop normally and express normal levels of the three CXCL12 isoforms. Following induced acute ischemia, a markedly impaired capacity to support revascularization was observed associated with a reduced number of infiltrating cells in the ischemic tissue. Importantly, exogenous administration of CXCL12gamma, which binds HS with the highest affinity ever reported for a cytokine, fully restores vascular growth, while HS-binding CXCL12gamma mutants failed to promote revascularization in Cxcl12gagtm/gagtm animals. These findings, recently reported, prove the role played by HS-interactions in the functions of CXCL12, both in homeostasis and physiopathological settings and document originally the paradigm of chemokine-immobilization in vivo. Based in these recent findings, the new proposal will explore the contribution of proteoglycan/CXCL12 interactions in the regulation of immune responses. Furthermore, we will extend our research to other constitutive chemokines involved in the regulation of migration and tissue homing of leukocytes.
We have recently discovered that Cxcl12gagtm/gagtm mice display reduced serum levels of gammaglobulins. In addition, analysis of B cell subpopulations in the mutant mice revealed a consistent reduction in the number of recirculating mature B cells and plasma cells in the BM. These original observations strongly support a role for CXCL12/HS interactions in the fine-tuning of the homeostasis in the B cell compartment. The mechanisms underlying this anomaly will be investigated in this project. Our hypothesis is that the lack of CXCL12-HS binding affects the correct trafficking and retention of activated B cells in homeostatic and immunized conditions. Furthermore, we believe that the example provided by CXCL12 is a paradigm of the contribution of HS, or other GAG, to the biological function of other constitutive chemokines, such as CXCL13, CCL21 or CCL19, which are spatially and functionally related to CXCL12 in the control of migration and homing of leukocytes and the organization of secondary lymphoid organs. The lack of structural and biochemical information on the interactions of these chemokines with GAG structures limits the ability to perform in vivo analysis, as relevant as those carried out for CXCL12. This has deprived us of a complete picture of the contribution of this elusive function to the homeostasis of secondary lymphoid organ organization and immune orchestration. The aim of this proposal is to fill in this gap in knowledge. We believe that the knowledge and experience accumulated during previous research and the ongoing collaboration set up by the current partnership that gathers scientist expert in chemokine biochemistry and glycobiology, B/T lymphocyte biology and dynamic imaging of immune cells warrants the feasibility of this project.