Ex vivo characterization of human thymic dendritic cell subsets
Introduction
Thymic dendritic cells (DC) have specific functions compared to blood DC. Indeed they can present self-antigens and induce negative selection, the induction of apoptotic death in potentially self-reacting developing T-cells (Brocker et al., 1997). They may also be involved in additional tolerogenic mechanisms like the induction of regulatory T-cells. Human thymus contains distinct thymic DC populations. Human thymic CD11c+ DC are defined phenotypically as HLA-DR+ CD11c+ CD4+ CD45RAlow cells. However, the description of the different human thymic CD11c+ DC subsets varies depending on the isolation procedure used (Bendriss-Vermare et al., 2001; Res et al., 1999; Schmitt et al., 2000; Vandenabeele et al., 2001). Two populations of CD11c+ CD14− DC with morphological and phenotypical differences have been described: one HLA-DRint CD83− CD86low DC-Lamp− and the other HLA-DRhigh CD83+ CD86high DC-Lamp+ (Bendriss-Vermare et al., 2001). Thymic CD14+ DC were also described according to their phenotypes and functions (Vandenabeele et al., 2001). This paper described two populations of CD11c+ DC: CD11b− CD14− and CD11b+ CD14+. Studies on human thymic DC development suggest that, as in the mouse, many CD11c+ DC derive from an intrathymic lymphoid-restricted pathway (Marquez et al., 1998; Res et al., 1996). Some human thymic DC may also develop through a myeloid pathway (de Yebenes et al., 2002).
Human thymus also contains plasmacytoid DC (pDC) sharing common functional properties with blood and secondary lymphoid organs pDC (Bendriss-Vermare et al., 2001; Res et al., 1999; Schmitt et al., 2000; Vandenabeele et al., 2001). However, thymic pDC express higher levels of CD2, CD5 and CD7 than their peripheral counterparts (Res et al., 1999). Thymic pDC can be distinguished from CD11c+ DC as being HLA-DR+ CD45RA+ CD11c− and CD123high. Thymic pDC also express lymphoid-related RNA transcripts, including pre-Tα and Spi-B (Bendriss-Vermare et al., 2001; Res et al., 1999).
Upon activation with IL-3 and CD40L, human thymic pDC have been shown to differentiate in culture into mature CD11c+ CD14− DC expressing CD83 and co-stimulatory molecules CD80 and CD86 at high levels (Bendriss-Vermare et al., 2001; Res et al., 1999). However, these mature DC have not yet been identified in vivo. In the absence of CD40 stimulation, thymic pDC also acquire CD11c but express CD86 at low levels (Bendriss-Vermare et al., 2001).
In the present study, we have used an approach that allows the isolation of a sufficient number of human thymic DC, permitting thorough characterization of their phenotypes and further study of their functions. We discriminated three major thymic DC populations based on CD123, CD11c and CD14 expression. In thymic pDC cultures supplemented with IL-3, the development of CD11c+ cells expressing low level of co-stimulatory molecules could be observed. Interestingly, these culture conditions led to the appearance of cells expressing CD11c as well as CD14, properties similar to freshly isolated human thymic CD11c+ DC.
Section snippets
Thymuses
Fresh thymus fragments were obtained during elective cardiac surgery (Hôpital Necker, Paris, France and Marie Lannelongue, Le Plessis Robinsson, France) on children (age range, 6 days–24 months). As human thymi are surgical wastes according to the French law regulating the usage of human tissues, informed consent from individuals was not required.
Isolation of thymic DC subpopulations
Cells were dissociated by gentle teasing. Non-dispersed cells were incubated with collagenase IV (5 mg/ml, Sigma, St. Louis, MO) and DNase (150 U/ml,
Identification of three thymic DC populations
To identify different thymic DC populations, we first studied the expression of DC markers on thymic cells. Percoll cell fractions were analyzed for surface expression of the thymic DC markers (Bendriss-Vermare et al., 2001; Vandenabeele et al., 2001). Cells were subject to triple staining for CD11c, CD123 and CD14 and then analyzed by flow cytometry. Distinct subsets expressing these molecules could be detected in the low-density cell fraction (Fig. 1). Cells expressing high levels of CD123
Discussion
In this study, we used a new method for the isolation of whole DC populations to define three thymic DC subsets on the basis of CD123, CD11c and CD14 expression (pDC, CD11c+ CD14− DC and CD11c+ CD14+ DC) and determine their phenotypes and anatomical locations. We have also shown that CD123low/− CD11c+ CD14+ cells could be detected in pDC cultures supplemented with IL-3. Interestingly, these cells expressed low levels of maturation molecules and presented a phenotype similar to that of freshly
Acknowledgments
We thank Dr. Sonia Berrih-Aknin (Hôpital Marie Lannelongue, Le Plessis-Robinson, France) and Professor Leca (Hôpital Necker, Paris, France) for providing us with thymi. We thank Gianfranco Pancino, Anne Hosmalin and Matthew Albert for the critical reading of the manuscript and helpful discussion, and Francine Brière for useful information. We thank Gerard Zurawski and Kimberly Gehlbach for proofreading the manuscript. N. Schmitt was successively the recipient of fellowships from the French
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2020, ImmunityCitation Excerpt :P2 cells were not flagged as doublets in a Scrublet analysis (Figure S3C) and showed similar UMI counts as P3 cells (mean UMI per cell = 9,988–19,461 versus 9,403–22,422, P2 versus P3 for thymuses 1–3, p > 0.1), making them unlikely to be doublets. P2 showed very little expression of PAX5 (expressed in 7% of cells) and CD19 (5% of cells) (Figure 3B) and did not express the mature pDC markers NRP1 (Collin and Bigley, 2018) or CD4 (Schmitt et al., 2007), making it unlikely to represent a B lineage committed or mature pDC population (Medvedovic et al., 2011). P2 also showed relatively lower expression of HSPC genes than other early clusters (P1 and P3) (Figure 1C), further supporting it as being transcriptionally distinct from other uncommitted thymic progenitor populations.
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2012, Clinical ImmunologyCitation Excerpt :Furthermore, in a given tonsil, follicles were either all positive, to a different extent, or strikingly all negative. Staining with various DC markers revealed that CD11c+CD13+ DC, which have previously been described in the thymus [43] and tonsils [44,45] appeared to be responsible for follicular LL-37 expression. Isolation of these cells showed that most stained strongly for LL-37 whereas a few were negative or weakly positive for the peptide (Fig. 5e).
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2009, BloodCitation Excerpt :Our current work, which demonstrated thymoproteasomes in human cTECs, indicates that they may also be involved in positive selection of human T cells. In the human thymus, several discrete populations of DCs, such as immature myeloid, mature myeloid, and plasmacytoid DCs, have been identified using combinations of phenotypic markers.16,17 It is widely accepted that thymic DCs are largely distributed in the medulla and play a crucial role in the process of negative selection.18
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This work is dedicated to the memory of Nicole Israel.