CD69

 

Description

CD69 is a membrane‐bound, type II C‐lectin receptor, usually identified as a classical early marker of lymphocyte activation, due to its rapid appearance on the surface of the plasma membrane after stimulation. CD69 is expressed by several subsets of tissue resident immune cells, including resident memory T (TRM) cells and gamma delta (gd) T cells, and is therefore considered a marker of tissue retention. Recent evidence has revealed that CD69 regulates the differentiation of regulatory T cells (Treg) as well as the secretion of IFN‐g, IL‐17, and IL‐22 (Cibrian and Sanchez-Madrid 2017).

 

Alignment

Protein alignment for human, rhesus macaque and marmoset CD69:

The referenced media source is missing and needs to be re-embedded.

 

Protein alignment for marmoset, owl monkey, and squirrel monkey CD69:

The referenced media source is missing and needs to be re-embedded.

 

References

  • Cibrian, D. and F. Sanchez-Madrid (2017). "CD69: from activation marker to metabolic gatekeeper." Eur J Immunol 47(6): 946-953.

 

Status

The extracellular domain of marmoset CD69 was produced in HEK239 cells, with a C-tag and an immunogenicity tag. After immunization of mice with this antigen, no antibodies were detected when using the soluble Twin-tagged version of the molecule, but good responses were seen when coating ELISA plates with the same antigen. A total of two clones were selected after screening using ELISA, FluoroSpot (for clonality analysis) and Octet (for affinity scouting and epitope binning) (Figure 1). 

The two mAbs were grown and purified and tested in a flow cytometry assay using marmoset PBMC unstimulated or stimulated with SEB. The presence of the mAb on the surface of the marmoset PBMC was detected with a PE-labeled goat polyclonal antibody to mouse IgG. None of the two antibodies had detectable reactivity. With the possibility that the generated mAbs targeted a non-native version of CD69, a second assay was performed in which cells were fixed with formaldehyde and then tested with the same mAbs. Again, no reactivity was observed for either mAb. Controls for this experiment included a mouse mAb specific for CD28, which was identified on the surface of unstimulated and stimulated marmoset PBMC (data not shown).

NWMIR-Identification of mAbs against recombinant MAR CD69.
Figure 1. Identification of mAbs against recombinant MAR CD69. Hybridomas were screened using ELISA plates coated with the immunizing antigen; the numbers shown in the left table are OD values. Results of the epitope identification are shown on the right.

In summary, the two mAbs produced were able to bind to the immunizing recombinant MAR CD69 antigen, but did not bind to the native MAR CD69. We will repeat the immunization with SP2/0 cells expressing transmembrane CD69, which should induce immune responses targeting the native form of MAR CD69.