Rational design of synthetic peptides to generate antibodies that recognize in situ CD11c(+) putative dendritic cells in horse lymph nodes.

The research paper discusses how synthetic peptides were used to create antibodies that could identify CD11c(+) dendritic cells in horse lymph nodes. The researchers used a 3D model to select segments for peptide synthesis, generating murine polyclonal antisera against these peptides, and testing them in cell suspensions and lymph nodes.

Three Dimensional Model of Integrin CD11c

• The scientists used a three-dimensional model of the alphaX I-domain of a specific protein (integrin CD11c) that is found in dendritic cells, which are a type of immune cell.
• This protein is located on the surface of several types of immune cells including dendritic cells and plays an important role in cell adhesion, a process by which cells interact and attach to neighbouring cells.

Synthetic Peptides

• The model helped them to identify two specific segments of the protein’s primary structure that would be suitable for the creation of synthetic peptides.
• These peptides each contain a specific sequence of amino acids – Peptide 1 contains 20 amino acids and spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, and Peptide 2 has 17 amino acid residues going from Asp158 to Phe174, corresponding to an exposed segment of the loop considered to be the metal ion-dependent adhesion site.

Generation and Testing of Murine Polyclonal Antisera

• They then generated murine (mouse) polyclonal antisera against both of these synthetic peptides. Antisera refers to blood serum containing antibodies against specific antigens, used to pass on immunity to many diseases.
• These antisera were then tested in peripheral blood cell suspensions and in cryosections (tissue samples frozen for examination under a microscope) of horse lymph nodes.

Identifying Dendritic Cells

• Out of the two antisera, only the serum against Peptide 2 was successful in identifying cells both in suspension and when examined in situ (in their original place) by immunohistochemistry, a technique used to visualize and identify specific cellular components.
• In this process, they were able to identify a key immunogenic epitope (a part of an antigen that is recognised by the immune system) in the CD11c protein in cells from horse lymph nodes when examined in situ.
• Some of these identified cells had an evident dendritic morphology, implying that they were dendritic cells. These cells play a crucial role in initiating and shaping immune responses, therefore the identification of these cells could provide valuable insights into equine immune responses.