Ex vivo characterization of human thymic dendritic cell subsets

doi:10.1016/j.imbio.2007.01.008

Immunobiology

Volume 212, Issue 3, 15 May 2007, Pages 167-177

https://doi.org/10.1016/j.imbio.2007.01.008 Get rights and content

Abstract

Interactions between thymic dendritic cells (DC) and thymocytes are critical for proper development of T-cells. We identified human thymic DC populations on the basis of CD123, CD11c and CD14 expression. High levels of CD123 (IL-3R) and CD45RA defined the plasmacytoid DC (pDC) subset. Human thymic CD11c⁺ DC expressed CD45RO and myeloid-related markers (CD13, CD33 and CD11b). CD11c⁺ DC could be separated into two main subsets based on differential expression of CD14: CD11c⁺ CD14⁻ and CD11c⁺ CD14⁺ cells. Spontaneous production of IL-10 and IFNγ without exogenous stimulation, was observed in the three DC subsets. Important phenotype modifications were observed in pDC cultures supplemented with IL-3. A down-regulation of CD123 and appearance of myeloid markers such as CD11b and CD11c on CD45RA⁺ cells was noticed within the first 48 h; at a later time there was a shift from CD45RA to CD45RO expression, as well as appearance of CD14 expression. CD11c⁺ cells emerging in pDC culture did not express high levels of HLA-DR, CD83 and co-stimulatory molecules. This suggests an in vitro evolution of human thymic pDC toward a myeloid phenotype found in the CD11c⁺ subset of thymic DC.

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⁺ CD45RA^low 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-DR^int CD83⁻ CD86^low DC-Lamp⁻ and the other HLA-DR^high CD83⁺ CD86^high 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 CD123^high. 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 CD123^low/− 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

References (24)

V.G. de Yebenes et al.
Identification of a myeloid intrathymic pathway of dendritic cell development marked by expression of the granulocyte macrophage-colony-stimulating factor receptor
Blood
(2002)
O. Duramad et al.
IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences
Blood
(2003)
K. Fukuhara et al.
A study on CD45 isoform expression during T-cell development and selection events in the human thymus
Hum. Immunol.
(2002)
C. Marquez et al.
Identification of a common developmental pathway for thymic natural killer cells and dendritic cells
Blood
(1998)
P. Res et al.
CD34+CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells
Blood
(1996)
P.C. Res et al.
Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus
Blood
(1999)
Q. Sun et al.
Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist
Blood
(1996)
S. Vandenabeele et al.
Human thymus contains 2 distinct dendritic cell populations
Blood
(2001)
G.A. Waanders et al.
Treatment of fetal thymic organ culture with IL-1 leads to accelerated differentiation of subsets of CD4-CD8- cells
Cell Immunol.
(1992)
A. Zlotnik et al.
Cytokine production and requirements during T-cell development
Curr. Opin. Immunol.
(1995)

N. Bendriss-Vermare et al.

Human thymus contains IFN-alpha-producing CD11c(−), myeloid CD11c(+), and mature interdigitating dendritic cells

J. Clin. Invest.

(2001)

T. Brocker et al.

Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo

J. Exp. Med.

(1997)

Cited by (17)

Leptin regulates thymic plasmacytoid dendritic cell ability to influence the thymocyte distribution in vitro
2023, International Immunopharmacology
Leptin, the adipocyte-derived hormone, involved in regulating food intake and body weight, plays an important role in immunity and reproduction. Leptin signals via the specific membrane receptors expressed in most types of immune cells including dendritic cells (DCs) and thymocytes. Leptin enhances thymopoiesis and modulates T-cell-mediated immunity. Thymic plasmacytoid DCs (pDCs) are predominated in the thymus. They play an important role in thymocyte differentiation. We have analyzed whether leptin mediates its effects on human thymocytes by influencing on pDCs. We used leptin at concentration corresponding to its level during II-III trimesters of physiological pregnancy. We cultivated leptin-primed pDCs with autologous thymocytes and estimated the main thymocyte subsets expressing αβ chains of the T-cell receptor (αβTCR), natural regulatory T-cells (tTreg), natural T-helpers producing interleukin-17 (nTh17) and invariant natural killer T-cells (iNKT) in vitro. We have shown that leptin augmented CD86, CD276 expressions and depressed IL-10 productions by pDCs. Leptin-primed pDCs decreased the percentage of CD4⁺CD8⁺αβTCR⁺ thymocytes, increased CD4^hiCD8^−/loαβTCR⁺ cells. pDCs cultivated with leptin decreased the number of iNKT precursors, and did not change the number of tTreg and nTh17 precursors. Thus, leptin’s important role in regulation of thymic pDC abilities to influence on the thymocyte distribution was indicated in vitro.
Single-Cell RNA-Seq Mapping of Human Thymopoiesis Reveals Lineage Specification Trajectories and a Commitment Spectrum in T Cell Development
2020, Immunity
Citation 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.
The challenges in recapitulating in vivo human T cell development in laboratory models have posed a barrier to understanding human thymopoiesis. Here, we used single-cell RNA sequencing (sRNA-seq) to interrogate the rare CD34⁺ progenitor and the more differentiated CD34^– fractions in the human postnatal thymus. CD34⁺ thymic progenitors were comprised of a spectrum of specification and commitment states characterized by multilineage priming followed by gradual T cell commitment. The earliest progenitors in the differentiation trajectory were CD7^– and expressed a stem-cell-like transcriptional profile, but had also initiated T cell priming. Clustering analysis identified a CD34⁺ subpopulation primed for the plasmacytoid dendritic lineage, suggesting an intrathymic dendritic specification pathway. CD2 expression defined T cell commitment stages where loss of B cell potential preceded that of myeloid potential. These datasets delineate gene expression profiles spanning key differentiation events in human thymopoiesis and provide a resource for the further study of human T cell development.
Dendritic cells in the host response to implanted materials
2017, Seminars in Immunology
Citation Excerpt :
Thymic pDCs are similar in immunophenotype to the aforementioned peripheral blood pDCs. Mature CD11c+ mDCs make up about 7% of thymic DCs and are marked by their expression of DC-LAMP, CCR7 and IL-12 production [12]. Lymphoid tissues (e.g., spleen, lymph nodes) are also resident to significant numbers of DCs.
The role of dendritic cells (DCs) and their targeted manipulation in the body’s response to implanted materials is an important and developing area of investigation, and a large component of the emerging field of biomaterials-based immune engineering. The key position of DCs in the immune system, serving to bridge innate and adaptive immunity, is facilitated by rich diversity in type and function and places DCs as a critical mediator to biomaterials of both synthetic and natural origins. This review presents current views regarding DC biology and summarizes recent findings in DC responses to implanted biomaterials. Based on these findings, there is promise that the directed programming of application-specific DC responses to biomaterials can become a reality, enabling and enhancing applications almost as diverse as the larger field of biomaterials itself.
The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils where it is exclusively expressed by neutrophils and a subset of dendritic cells
2012, Clinical Immunology
Citation 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).
Antimicrobial peptides are essential elements of epithelial defense against invading micro-organisms. The palatine tonsils are positioned at the entry of the airway and the gut and as such are ideally situated to act as immune sentinels in the pharynx protecting against microbial invasion. Tonsils express a number of antimicrobial peptides including hCAP18/LL-37. Here we clearly define the expression of hCAP18/LL-37 in the tonsils showing unequivocally that hCAP18/LL-37 is mainly expressed by infiltrating neutrophils and follicular CD11c+CD13+HLA-DR+ dendritic cells, rarely by macrophages, and never by the epithelium itself. To explore possible functions for follicle-derived LL-37, we stimulated tonsil mononuclear cells with LL-37 in vitro and observed the secretion of the proinflammatory cytokines CCL5 and CXCL9, expression of IFN-γ and MX-1 and down-regulation of chemokine receptors CCR4 and CCR6 which are involved in tissue-selective T cell trafficking. Taken together, these data illustrate new potential immunoregulatory functions for hCAP18/LL-37 in the tonsils.
Transformation of dendritic cells from plasmacytoid to myeloid in a leukemic plasmacytoid dendritic cell line (PMDC05)
2010, Leukemia Research
Citation Excerpt :
Zuniga et al. [35] reported that bone marrow pDCs could differentiate into mDCs upon viral stimulation by using viral-infected experiments with mice. Schmitt et al. [36] also reported that human thymic pDCs could differentiate into mDCs with IL-3 stimulation in vitro. Moreover, there was a report that the CD5+/CD56+ pDC subset is intermediate between pDC and mDC in peripheral blood pDC from an Flt3L-treated healthy volunteer [37].
We investigated the transformation from pDCs to mDCs in a pDC line (PMDC05) which was established from a patient with pDC leukemia in our laboratory. PMDC05 cells were separated into two fractions according to the expression of BDCA1 and CD123. BDCA1⁻CD123⁺ cells were found to be pDC-like cells by their morphology, surface phenotypes, mRNA expression and the function. In addition, BDCA1⁻CD123⁺ cells were demonstrated to have a proliferating capacity and revealed the ability to transform to BDCA1⁺CD123⁻ cells which showed mDC-like properties. Our data demonstrated the possibility of transformation from pDCs to mDCs in human DC lineage.
Exclusive expression of proteasome subunit β5t in the human thymic cortex
2009, Blood
Citation 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
The ubiquitin-proteasome pathway, which degrades intracellular proteins, is involved in numerous cellular processes, including the supply of immunocompetent peptides to the antigen presenting machinery. Proteolysis by proteasomes is conducted by three β subunits, β1, β2, and β5, of the 20S proteasome. Recently, a novel β subunit expressed exclusively in cortical thymic epithelial cells was discovered in mice. This subunit, designated β5t, is a component of the thymoproteasome, a specialized type of proteasomes implicated in thymic positive selection. In this study, we show that, like its mouse counterpart, human β5t is expressed exclusively in the thymic cortex. Human β5t was expressed in approximately 80% of cortical thymic epithelial cells and some cortical dendritic cells. Human β5t was incorporated into proteasomes with two other catalytically active β subunits β1i and β2i, forming 20S proteasomes with subunit compositions characteristic of thymoproteasomes. The present study demonstrates, for the first time, the existence of thymoproteasomes in the human thymic cortex, indicating that thymoproteasome function is likely conserved between humans and mice.

View all citing articles on Scopus

¹: This work is dedicated to the memory of Nicole Israel.

View full text

Ex vivo characterization of human thymic dendritic cell subsets

Abstract

Introduction

Section snippets

Thymuses

Isolation of thymic DC subpopulations

Identification of three thymic DC populations

Discussion

Acknowledgments

Blood

Blood

Hum. Immunol.

Blood

Blood

Blood

Blood

Blood

Cell Immunol.

Curr. Opin. Immunol.

Human thymus contains IFN-alpha-producing CD11c(−), myeloid CD11c(+), and mature interdigitating dendritic cells

J. Clin. Invest.

Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo

J. Exp. Med.