Nevertheless, a few exceptions were seen. obtained. Rabbit Polyclonal to ARC Dendritic cells (DC) in lymphoid and non-lymphoid tissues are professional antigen-presenting cells that are necessary for pathogen recognition and the initiation of primary T-cell immune responses.1, 2 Although DC in the thymus may also have a role in the protection against certain infections, it is the presentation of self-antigens to the developing T-cell pool that makes DC indispensable for the proper establishment of central tolerance. Thymic DC were not only shown to promote unfavorable selection of autoreactive T cells, but may also participate in the induction of regulatory T cells (reviewed in Klein em et al. /em 3 and Hadeiba and Butcher4). As described for various peripheral organs, human thymic DC are most likely heterogeneous regarding their origin and ontogeny, anatomical localization, capacity to take up and process different forms of antigen, Mps1-IN-3 and production of various cytokines. It can be anticipated that these differences influence the responses of the maturing T cells interacting with them. Some of these functional differences of thymic DC populations will be reflected by a differential expression of cell surface molecules. Similar to other tissues, human thymus contains two major types of DC: plasmacytoid DC (pDC, defined as HLA-DR+ cells expressing IL-3R (CD123), but lacking expression of CD11c) and conventional DC (cDC, expressing CD11c and high amounts of HLA-DR).5, 6 Within thymic cDC, differential expression of several cell surface molecules, like DC-LAMP, CD14 or CD11b, pointed to the existence of two different subsets also in the human thymus.5, 6, 7 However, an unequivocal classification of thymic cDC was lacking because of the absence of highly specific markers. To be able to consistently classify human thymic cDC, we compared a number of surface molecules used in the past for cDC subset definition in the peripheral blood or tissues, including CD141 Mps1-IN-3 (BDCA-3, thrombomodulin) and CD1c (BDCA-1).8, 9, 10 In these comparisons, we also included molecules that specifically describe functionally different cDC subsets in various mouse tissues, namely XCR1 and SIRP.11, 12 These comparisons revealed that human thymus also contains CD141+ cDC, which perfectly match the described CD11bneg thymic cDC.7 CD141+ CD11bneg thymic cDC express XCR1, but lack SIRP (13and data not shown) and thus resemble cross-presenting’ DC in peripheral blood.14, 15 Further, we found that all human thymic CD11b+ cDC co-express SIRP, and lack XCR1 expression, and thus phenotypically resemble human peripheral CD1c+ cDC (13and data not shown). Thus, human thymic cDC can consistently be subdivided into two subsets: CD141+ cDC and SIRP+ cDC (which are congruent with CD11b+ cDC). For practical reason (monoclonal antibody (mAb) availability, greater flexibility for combination with various HLDA10 mAb formats), CD11b (and not SIRP) together with CD141 was used for cDC classification in this study. The panel of 71 monoclonal antibodies from the 10th Human Leukocyte Differentiation Antigens workshop (HLDA10) was investigated for binding to freshly isolated human thymic pDC, CD141+ cDC or CD11b+ cDC. In this report, the results obtained are summarized. Results Because of the low abundance of DC in thymic tissue (approximately 0.2C0.3%), DC were enriched from digested human thymi by Nycodenz-gradient centrifugation to 1C4% pDC (defined as live CD123+ HLA-DR+ cells) Mps1-IN-3 and 6C10% cDC (defined as live CD123neg lineageneg HLA-DR+ CD11cint/high cells). The cDC were further subdivided into CD141+ or CD11b+ cDC (Physique 1). This enrichment allowed an efficient simultaneous analysis of all primary thymic DC subsets. At the same time, gating on lineageneg HLA-DR+ CD11cneg CD141neg CD123neg cells allowed to analyze other HLA-DR+ thymic cells, which were not DC (HLA-DR+ non-DC). Gating on HLA-DRneg, but lineage-positive cells encompassed mainly immature T cells, but also CD56+ NK cells. The results, summarized in Table 1, were highly reproducible, with each of the HLDA10 mAb giving comparable staining profiles on cells of all tested donors. Slight variations between single donors were observed only in the absolute fluorescence intensities or in the.
