Expression analysis of surface molecules on human thymic dendritic cells with the 10th HLDA Workshop antibody panel

Abstract

Dendritic cells (DC) in the thymus have an important role in the establishment of central tolerance by promoting negative selection of autoreactive T cells and regulatory T-cell differentiation. Whereas human DC have recently been studied in various tissues in more detail, thymic DC subsets are still ill-defined. In the present work, we studied the binding of 71 monoclonal antibodies (mAb) submitted to the HLDA10 workshop to human CD123+ plasmacytoid DC and the two subsets of conventional DC (cDC, CD141+ and CD11b+) isolated from thymus tissue of infants undergoing corrective heart surgery. Within the panel, we found mAb binding to thymic pDC and both cDC subsets (for example, anti-Clec12A, TIM-3, Clec4A, CCR5, Axl, FLT3), but most of them additionally reacted with other thymic cell types. MAb directed to CD85h (ILT1) and the C-type lectin Clec7A (now CD369) reacted selectively with both cDC subsets, but not with other cells. Only one mAb directed to CD85g (ILT7) stained thymic pDC in a highly specific manner. Clec9A (DNGR1, now CD370) was the only tested HLDA10 antigen exclusively expressed on thymic CD141+ cDC. The present report summarizes all data obtained.

Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP)

“…Control cells were treated with DMSO carrier. Anti-LPAP mAbs CL3 and CL7 were produced in our laboratory [ 25 ] and their specificity were tested on the 10 th HLDA [ 26 ]. Anti-tubulin mAb (12G10) was purchased from The Developmental Studies Hybridoma Bank.…”

“…There are several identified LPAP phosphorylation sites [ 15 – 24 ], but these data were gleaned from bulk large-scale proteomic studies, and, unfortunately, do not specify stoichiometry or functional significance of particular phosphosites. A new set of anti-LPAP monoclonal antibodies, generated in our laboratory [ 25 ] and tested on the 10 th human leukocyte differentiation antigen (HLDA) [ 26 ], provide an additional instrument for LPAP investigation. Using these antibodies, we identified at least five different LPAP phosphoforms in lymphocytes [ 27 ].…”

Autoimmune risk variants in ERAP2 are associated with gene-expression levels in thymus

“…Both conventional DCs (cDCs) and plasmacytoid DCs (pDCs) are found in thymus, where the cDCs constitute around two-thirds of the thymic DC population. The major subset of cDCs (roughly 2/3) can further be classified as CD8α+SIRPα-cDCs in mice [72] or CD141+ in humans [82]. The CD8+ SIRPα-cDCs are commonly referred to as the resident cDCs, as they arise from an intrathymic differentiation pathway [72].…”

“…There is also accruing evidence that these cDCs can present TRAs that have been transferred by mTECs, although the direct handover has been technically challenging to prove [72]. The minor subset of cDCs in thymus (roughly 1/3) can be characterized as CD8-SIRPα+ cDCs in mice and CD11b+ in humans [82,83]. These are migratory cDCs which are enriched in cortico-medullary perivascular space and also around small vessels, where they effectively capture and present blood borne antigens [84].…”

“…These are migratory cDCs which are enriched in cortico-medullary perivascular space and also around small vessels, where they effectively capture and present blood borne antigens [84]. Finally, the last DC type present in both mouse and human thymus are the pDCs (characterized as CD123+ in humans [82]). These are also migratory DCs, which capture and transport peptides from the blood stream into the thymus, where they present the peptides to the SP thymocytes [85].…”