Visualization of specific activated DCs

Data characterizing the antigen-presenting DC in immunized mice is available.

Our goal was to generate a probe that could allow the visualization of specific activated DCs following either the immunization of mice with an antigen in adjuvant or an infection with leishmania major parasites. We thus tried to prepare a mAb reacting to the immunodominant LACK156-173 peptide of leishmania bound to I-Ad MHC class II molecules. To this aim, we injected I-Ad/LACK recombinant dimers to TCR transgenic mice which exhibited an increased frequency of LACK-specific T cells. Four out of 600 supernatants, i.e. 2C44, 2F74, 2E60 and 2X8, readily stained LACK156-173-pulsed fibroblasts, but neither unpulsed fibroblasts nor OVA323-339-pulsed cells. BIAcore measurements yielded equilibrium dissociation constants ranging from 1.1 nM for 2C44 mAb to 120 nM for 2F74 mAb. To investigate whether these mAb could be used to detect APC presenting LACK in vivo, BALB/c mice were immunized or not with either LACK or OVA. LN cells were purified 2 days later, and stained with either 2C44, 2F74, 2E60 or 2X8 mAb. None of these mAb stained DC purified from non immunized mice. However, among the 4 mAb, only 2C44 could stain DC in LACK-immunized mice, but not in OVA-immunized mice. Furthermore, 2C44 stained DC from LACK-immunized mice expressing the H2-d haplotype but did not stain DC from LACK-immunized mice of other haplotypes. Thus, the 2C44 mAb allows for the detection of DC which captured and processed LACK in vivo. We next attempted to characterize the DC which carried I-Ad/LACK complexes at the cell surface in LACK-immunized BALB/c mice. Two different populations of DC, could thus be identified both expressing high levels of CD11c and MHC class II complexes but different levels of CD11b, suggesting that both Langerhans cells and dermal DC were actually presenting the LACK peptide. Following an intradermal immunization with LACK in adjuvant. We finally tried to purify these cells by cell sorting, using 2C44 mAb. In preliminary experiments we succeeded in purifying 300 000 cells from 5 immunized mice.
Having characterized the antigen-presenting DC in immunized mice, we have now started to visualize and characterize the DC which carry I-Ad/LACK complexes at the cell surface in BALB/c mice infected by leishmania major.

• stimulus type
• Leishmania major

• organ type
• skin

• molecule type
• Major histocompatibility complex class II,
• Ovalbumin,
• Leishmania LACK protein

• cell type
• fibroblast,
• T cell,
• dendritic cell

• organism type
• Mus musculus

created over 15 years ago (2 March 2009)    last modified over 12 years ago (3 September 2012)   [ RDF ]   [ RelFinder ]