Part:BBa_K1993013

CXCR5

Chemokine receptors are receptors found on the surface of certain cells that interact with chemokines. They have a 7-transmembrane (7-TM) structure and couple to G-protein for signal transduction within a cell. [1] (Figure 1) Following interaction with their specific chemokine ligands, chemokine receptors trigger a flux intracellular calcium (Ca2+) ions, initiate chemotaxis and guide the cell to a desired location. (Figure 2)

Figure 1. typical structure of a chemokine receptor.

Figure 2. the mechanism of interaction between a chemokine and a chemokine receptor.

Under the circumstances of inflammation, various kinds of cytokines, including chemokines, are released by the lesions. Guided by the chemokines, cells expressing chemokine receptors move towards the lesions where they can function better.[2] What’s more, different diseases would release different pools of chemokines, which would recruit different effector cells. See our disease table-chemokine

Based on the chemotaxis theory, in order to enhance the homing ability of our marvelous mesenchymal stem cells (MSCs) due to lack of enough chemokine receptors on their cell surface, we, SYSU-MEDICINE, had constructed a series of chemokine receptors that corresponding to different inflammatory diseases as far as possible. Among which, CXCL13 is a significant chemokine (CXCR5 is its chemokine receptor) in Delayed Type Hypersensitivity(DTH). See our disease table-chemokine

We acquired this gene from peripheral mononuclear blood cells (PMBCs) and purified it. (Figure 3) Then we constructed it under the control of EF-1α by Gateway technology. (Figure 4)

Figure 3 Purification of CXCR5

Figure 4 EF-1α-CXCR5

We introduced that plasmid into MSCs and tested the expression of CXCR5 in MSCs on mRNA and protein level (Figure 5, Figure 6)

Figure 5. Relative mRNA Level of CXCR5.

Figure 6. Western Blot of CXCR5.

Then, we tested the chemotaxis of engineered MSCs by conducting transwell assay against CXCL13. To our excitement, our engineered MSCs had improved their homing ability with chemokine receptor CXCR5(Figure 7,Figure8).

Figure 7,Figure 8. Transwell Assay of CXCR5.

MIT_MAHE 2020

Summary

The homeostatic receptors CXCR5 and CCR7 and their ligands are major regulators of the immune response. They act at the level of B and T lymphocytes and DCs trafficking to and within secondary lymphoid tissue. The presence of CXCR5+ T cells in peripheral blood reflects ongoing humoral immune responses, CXCR5 may even serve as a unique and convenient biomarker for the evaluation of ongoing vaccination responses. CXCR5+ T cells were relatively scarce in peripheral blood of healthy individuals.

References

[1]Allen, Samantha J.; Crown, Susan E.; Handel, Tracy M. (2007-01-01). "Chemokine: receptor structure, interactions, and antagonism". Annual Review of Immunology. 25: 787–820.

[2] Griffith J W, Sokol C L, Luster A D. Chemokines and chemokine receptors: positioning cells for host defense and immunity.[J]. Annual Review of Immunology, 2014, 32(1):659-702.

[3] Allen, C. D., Ansel, K. M., Low, C., Lesley, R., Tamamura, H., Fujii, N., & Cyster, J. G. (2004). Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nature immunology, 5(9), 943–952. https://doi.org/10.1038/ni1100

[4]Moser B. (2015). CXCR5, the Defining Marker for Follicular B Helper T (TFH) Cells. Frontiers in immunology, 6, 296. https://doi.org/10.3389/fimmu.2015.00296

[5] Barella, L., Loetscher, M., Tobler, A., Baggiolini, M., & Moser, B. (1995). Sequence variation of a novel heptahelical leucocyte receptor through alternative transcript formation. The Biochemical journal, 309 ( Pt 3)(Pt 3), 773–779. https://doi.org/10.1042/bj3090773

[6] Schaerli, P., Loetscher, P., & Moser, B. (2001). Cutting edge: induction of follicular homing precedes effector Th cell development. Journal of immunology (Baltimore, Md. : 1950), 167(11), 6082–6086. https://doi.org/10.4049/jimmunol.167.11.6082

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 31
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 973
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 259
  Illegal SapI site found at 1050