The precise lineage of dendritic cells (DCs), including skin Langerhans cells (LCs), is unclear. Interleukin 7 (IL-7) and its receptor (IL-7Rα) are known to mediate lymphopoiesis, and IL-7 is also known to be essential for the generation of DCs from lymphoid-committed precursors in vitro. Thus, to determine the developmental lymphoid (or IL-7Rα) dependency of various DCs and to examine the importance of IL-7/IL-7Rα for DC development in vivo, we used IL-7Rα knockout (KO) donor cells to reconstitute DCs/LCs in sublethally irradiated recipients and compared the results to those obtained using wild-type (WT) donor cells. We found that lymphoid lineage cells (except natural killer [NK] cells), including thymocytes, were less efficiently reconstituted by IL-7Rα KO donor cells, whereas myeloid lineage cells and DCs/LCs were equally well reconstituted by both the IL-7Rα KO and WT donor cells. Overall, we conclude that IL-7Rα is not required for the development of DCs/LC in vivo.
The precise lineage of dendritic cells (DCs), including epidermal Langerhans cells (LCs), is unclear.1 Because of their many similarities to macrophages, DCs (and LCs) were thought to be of myeloid lineage and were generated from monocytes.2 Considerable evidence also suggests a lymphoid lineage because CD8+ thymic DCs can be generated from CD4low early thymic precursors in vivo.3 More recent studies have demonstrated the generation of both CD8- and CD8+ DCs from either myeloid- or lymphoid-committed progenitors.4,5
Interleukin 7 (IL-7) plays a critical role in lymphopoiesis6-8 and has been shown to be important for DC generation from lymphoid-committed cells in vitro.9-11 Many in vitro studies have suggested that LCs are of myeloid lineage12,13 ; however, thymic precursor cell populations have been reported to be LC precursors in vivo.14 Because some thymic precursor populations are known to express IL-7Rα,15,16 IL-7 may be involved in the generation of LCs as well. Therefore, if we equate “lymphoid lineage” with “IL-7/IL-7Rα-dependent” then DC/LC lineages can be directly assessed using bone marrow (BM) from IL-7Rα knockout (KO) mice as donor cells to reconstitute various types of DCs including LCs in sublethally irradiated recipient mice, where some host cells are expected to survive. We used a BM-reconstitution x-irradiated mouse model where the sublethal irradiation dose is high (7.6 Gy); donor-dominant cellular chimerism is expected when wild-type (WT) donor cells are used, whereas when IL-7Rα KO donor cells are used, host-dominant lymphoid chimerism is expected because of the inability of the transferred IL-7Rα KO donor cells to develop as lymphoid lineage cells.
Reagents, monoclonal antibodies, and kits
All monoclonal antibodies (mAbs) were purchased from BD Bioscience PharMingen (San Diego, CA) except for the anti-PDCA-1 antibody, which was purchased from Miltenyi Biotec (Auburn, CA). Propidium iodide (PI), deoxyribonuclease I (DNase I), ammonium thiocyanate, and olive oil were from Sigma-Aldrich (St Louis, MO); trinitrochlorobenzene (TNCB) was from Polyscience (Warrington, PA); 1 × phosphate-buffered saline (PBS) and 1 × Hanks balanced salt solution (HBSS) were from Invitrogen (Grand Island, NY); fetal bovine serum (FBS) was from Gemini Bio-Products (Woodland, CA); trypsin was from USB (Cleveland, OH); and ACK lysing buffer and 0.5 M EDTA were from Quality Biological (Gaithersburg, MD).
Animals and BM reconstitution protocol
IL-7Rα KO mice (C57BL/6 background: CD45.2) were purchased from the Jackson Laboratory (Bar Harbor, ME) and C57BL/6 (WT control: CD45.2) and B6-Ly5.2/Cr (recipient: CD45.1) mice from the NCI/Frederick Cancer Research and Development Center (FCRDC; Frederick, MD). Recipient mice were x-irradiated at a dose of 7.6 Gy 20 to 24 hours prior to intravenous transfer of 1 × 107 BM cells/recipient. One ear of each recipient mouse was painted with 20 μL of a 1.5% TNCB solution (in 4:1 of acetone-olive oil) immediately after cell transfer to enhance LC engraftment. The other ear was left unpainted. Recipient BM, thymi, spleens, peripheral lymph nodes (pLNs; mixture of axillary and superficial inguinal LNs, hereafter called pLNs), and epidermis were assessed for chimerism of various cell types 4 and 8 weeks after reconstitution with IL-7Rα KO or WT donor BM cells.
Preparation of single-cell suspensions from various lymphoid organs and skin, and of epidermal sheets
BM cells were collected by flushing mouse tibia and fibulae with HBSS media supplemented with 5% fetal calf serum (FCS). Spleen, thymus, or pLNs were mashed on a 100-μm mesh spleen filter, suspended in HBSS containing 5% FCS and treated with ACK lysing buffer. Epidermal-cell suspensions and epidermal sheets were prepared as previously described.17 All cell suspensions prepared from recipient BM, thymus, spleen, pLNs, or epidermis were washed with PBS with 5% FCS containing 2 mM EDTA once before use. For flow cytometric analysis, cells were stained with various antibodies for 20 minutes at 4°C after Fc blocking and were assessed using a Becton Dickinson FACS Calibur flow cytometer and analyzed using CELLQuest software version 3.3 (San Jose, CA).
Mean values and standard deviation (SD) calculation, graph production, and statistical evaluation (Student t test with 2-tailed and 2 sample unequal distribution method) were performed using CA-Cricket Graph III version 1.5.3 (Computer Associates International, Islandia, NY) and Microsoft Excel X for Mac (Microsoft, Seattle, WA). P values of less than .05 were considered significant.
Results and discussion
IL-7Rα KO cells reconstitute various DC subsets
To determine the extent of chimerism at 4 and 8 weeks by WT or IL-7Rα KO donor cells, flow cytometric analysis was performed using a combination of antibodies with donor (CD45.2) or host (CD45.1) cell markers (Supplemental Figure S1, available at the Blood website; click on the Supplemental Figures link at the top of the online article). All the myeloid lineage cells such as polymor-phonuclear (PMN) cells or monocytes in BM and spleen were well reconstituted, whereas lymphoid lineage cells, such as T cells, B cells, thymocytes (except for natural killer [NK] cells and thymic precursor populations) were poorly reconstituted by IL-7Rα KO donors, as compared to those reconstituted by WT donor cells. Various splenic and LN-DC and BM-plasmacytoid DCs18 were examined using the DC marker CD11c along with CD11b, CD8α, or B220 mAbs (Supplemental Figure S2). IL-7Rα KO donor cells reconstituted these populations as well as did the WT donor cells (Figure 1).
In another set of experiments, CD8α+ or CD11b+ thymic DCs and pLN, splenic, and thymic-plasmacytoid DCs were also examined using a more specific anti-PDCA-1 mAb (Miltenyi Biotec) and found to be reconstituted by IL-7Rα KO donor cells as well as by WT donor cells (Supplemental Figure S3).
IL-7Rα KO donor cells reconstitute LCs but not other epidermal leukocytes
IL-7Rα KO donor cells were able to give rise to LCs at 4 weeks after reconstitution as demonstrated in epidermal sheets prepared from irritated (TNCB-painted) ears (Figure 2A). No significant difference was found when donor cells came from IL-7Rα KO rather than from WT mice (Figure 2B and Supplemental Figure S3). In accordance with previous reports,19,20 epidermal γδ T cells were not reconstituted by either IL-7Rα KO or WT donor BM, even in irritated ears (Figure 2C). The major αβ T-cell population seen in irritated ears was of donor origin when WT donor cells were used and was of host (also WT) origin when IL-7Rα KO donor cells were used (Figure 2D). These αβ T cells are known to populate the epidermis of irradiated recipient mice,21,22 although usually at a later time period after reconstitution.
In the current study, lymphoid lineage cells (except for NK cells), including thymocytes, are less efficiently reconstituted by IL-7Rα KO donor cells as compared to WT donor cells, whereas myeloid lineage cells are well reconstituted by both the KO and WT donor cells, thereby validating this model. In recipient spleen, peripheral LNs and thymus, both CD8α+ and CD11b+ DC subsets are well reconstituted by both KO and WT donor cells at both 4 and 8 weeks after reconstitution, suggesting that IL-7Rα is not required for the development of these DCs in these organs and indicating that these DC subsets are not lymphoid dependent.
We also assessed the potential lymphoid (IL-7Rα) dependency of LCs. LCs are derived from BM23 and are renewed from within skin in the steady state and from BM when the steady state is disrupted.24 LC engraftment and the extent of LC chimerism did not differ when WT and IL-7Rα KO donor cells were compared, thus negating the possibility that LCs are lymphoid dependent.
Prepublished online as Blood First Edition Paper, September 15, 2005; DOI 10.1182/blood-2005-02-0647.
Supported by a research fellowship granted to S.T. by the National Cancer Institute (NCI).
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.
We thank Mr Jay Linton and Dr Fumiko Takeuchi for excellent technical help and Drs Mark Udey and Ronald E. Gress for helpful discussions.