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. 2011 Feb 1;186(3):1421-31.
doi: 10.4049/jimmunol.1002587. Epub 2010 Dec 22.

Functional Redundancy Between Thymic CD8α+ and Sirpα+ Conventional Dendritic Cells in Presentation of Blood-Derived Lysozyme by MHC Class II Proteins

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Functional Redundancy Between Thymic CD8α+ and Sirpα+ Conventional Dendritic Cells in Presentation of Blood-Derived Lysozyme by MHC Class II Proteins

Danielle F Atibalentja et al. J Immunol. .
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Abstract

We evaluated the presentation of blood-derived protein Ags by APCs in the thymus. Two conventional dendritic cells (cDCs), the CD8α(+)Sirpα(-)CD11c(hi) (CD8α(+) cDC) and the CD8α(-)Sirpα(+)CD11c(hi) (Sirpα(+) cDC), were previously identified as presenting MHC class II bound peptides from hen egg white lysozyme (HEL) injected intravenously. All thymic APCs acquired the injected HEL, with the plasmacytoid dendritic cell being the best, followed by the Sirpα(+) cDC and the CD8α(+) cDC. Both cDCs induced to similar extent negative selection and regulatory T cells in HEL TCR transgenic mice, indicating a redundant role of the two cDC subsets in the presentation of blood-borne HEL. Immature dendritic cells or plasmacytoid dendritic cells were considerably less efficient. Batf3(-/-) mice, with significantly reduced numbers of CD8α(+) cDCs, were not impaired in HEL presentation by I-A(k) molecules of thymic APCs. Lastly, clodronate liposome treatment of TCR transgenic mice depleted blood APCs including Sirpα(+) cDCs without affecting the number of thymic APCs. In such treated mice, there was no effect on negative selection or regulatory T cells in mice when administering HEL, indicating that the T cell responses were mediated primarily by the cDCs localized in the thymus.

Conflict of interest statement

The authors have no financial conflicts of interest.

Figures

Figure 1
Figure 1. All thymic DC subsets present HEL
HEL presentation was assayed on thymic DCs isolated from B10.BR mice. A) Shows the criteria for identifying the various DC subsets, based on expression of CD45RA, CD11c, CD8α and Sirpα. Post-sort indicates the purity of the DC sorted into Sirpα+ cDC (■), CD8α+cDC (▲), immature DC (▼) and pDC (◆). B) DC were isolated from B10.BR mice injected with 50µg HEL, 30min before. Cells were cultured for 24h with the 3A9 T-cell hybridoma, after which IL-2 was measured. C) Thymic DC isolated from un-injected mice were cultured with 3A9 T-cell hybridoma in the presence of continuous HEL, or D), with 10µM HEL 48–62 peptide. E) As in panel C, but HEL was pulsed for 1h and washed away. IL-2 production was measured by CTLL proliferation as indicated by [3H] thymidine incorporation. Data in B is representative of three experiments with each APC concentration tested in triplicate. Data in C–D and E is representative of two and four experiments respectively with each point or individual bar representing duplicate or triplicate samples. Error bars indicate SD.
Figure 2
Figure 2. Presentation by thymic cDC from Batf3−/− mice
A) DCs were isolated from B10.BR or Batf3−/− thymi. Left, gated DC subsets with pDC (CD45RAhiCD11cint), immature DC (CD45RAloCD11cint) and cDC (CD45RAloCD11chi). Right, Sirpα vs. CD8α expression gating on cDC. B) 3A9 T-cell hybridoma cultured for 24 hours with thymic CD11chi MHC II+ DC isolated and sorted from B10.BR mice (▲) or Batf3−/− mice (■) injected i.v. with 50µg HEL and sacrificed 30 min after injection. IL-2 production was measured by CTLL proliferation as indicated by [3H] thymidine incorporation. Data is representative of two experiments, with each APC concentration tested in triplicate. Error bars indicate SD.
Figure 3
Figure 3. Induction of thymocyte negative selection or activation by thymic DC subsets
A) Deletion of DPhi thymocytes in response to titrated DC from mice injected i.v. with 150µg HEL and isolated 30 min later. B) CD69 up-regulation on CD4SP thymocytes cultured with DC as in (A). C) (top), 3A9 thymocytes were cultured either alone (No Antigen) or with sorted DC subsets in the presence of 30µM HEL. (bottom) CD4SP T-cells were examined for CD69 up-regulation in response to culture with the DC. D) Deletion of DPhi thymocytes in response to titrated HEL when 3A9 thymocytes were cultured with sorted DC subsets. (E) CD69 upregulation on CD4SP cultured with DC as in (D). The DC subsets evaluated were Sirpα+ cDC (■), CD8α+cDC (▲), Immature DC (▼) and pDC (◆). Data is representative of two (Figures 3A–B) and six (Figures C–E) experiments with each antigen dose or APC concentration tested in duplicate or triplicate. Error bars indicate SD.
Figure 4
Figure 4. Treg induction to blood-borne HEL by thymic DC subsets
A) Percentage and B) absolute number of Foxp3+CD25+CD4+ cells induced after culture of Foxp3CD25CD4SP thymocytes from LB11.3 foxp3GFP mice with the indicated sorted DC subsets. APC were isolated and enriched from B10.BR mice injected i.v. with 50µg HEL 30min before (black bars) or un-injected (white bars). Data is representative of three experiments with error bars denoting SD.
Figure 5
Figure 5. Uptake of blood-borne HEL by intra-thymic cDC
(A) Uptake of HEL by thymocytes from B10.BR mice injected with saline or 500µg HEL HilytePlus™ 647. Mice were sacrificed at 5 min or 30 min post-injection. Graph shows the average percentage of HEL+ cells in the whole thymi fraction pooled from all the experiments with error bars indicating SEM. B) DC enriched fractions from mice injected with saline or HEL as in A. Left, total events of DC enriched fraction. Right, HEL fluorescence gating on total CD11c+ cells shown left: the saline injected, in solid gray, 5 min HEL in blue and 30 min HEL in green. C) Overlay of HEL Hilyte647 fluorescence by DC subsets gated on the DC enriched fraction shown in panel B. The numbers in each histogram represent the percentage of cells from the indicated subset that is positive for HEL and are from mice injected with saline (gray), HEL for 5 min (blue) or 30 min (green). Data is representative of three experiments, using DC isolated from seven (saline and 5 min) and eight mice (30 min).
Figure 6
Figure 6. Induction of negative selection by FLT3L DC expressing membrane bound HEL
A) Total cellularity, B) CD4SP and C) Foxp3+CD4SP cells in 3A9 mice injected with 2×107 FLT3L elicited DC from mHEL or B10.BR mice. Thymi were harvested 24 hours after injection. Graphs are from events gated on total lymphocytes with each bar representing pooled data from two experiments: B10.BR DC (7 mice), mHEL DC (11 mice). Statistical significance is indicated on graphs with (**), denoting p=0.0028, and (***) denoting p=0.006. Error bars indicate SEM.
Figure 7
Figure 7. HEL is rapidly acquired by both CD11c positive and negative subsets in the blood and cleared
A) Uptake of HEL HilytePlus™ 647 by blood CD11c+ and CD11c cells at different times post-injection. Percentage of HEL HilytePlus™ 647 staining by B) CD11c+ and C) CD11c cells in mice that were injected with HEL HilytePlus™ 647 (black bars) or saline (white bars) and sacrificed at the indicated times. D) (left), Sirpα and CD11c staining of peripheral blood cells from mice injected with saline (top) or HEL Hilyte647 (bottom) 1min after injection. (right) HEL uptake by CD11b positive and negative cells gating on the indicated subsets shown in the left panel. Data is representative of two experiments. Graphs are from events excluding red blood cells by SSC × FSC with each bar representing pooled data from two to three mice. Error bars indicate SD.
Figure 8
Figure 8. Liposome depletion of blood Sirpα+CD11c+ cDC does not impair negative selection and Tregs in response to HEL in the thymus
A) Depletion of blood Sirpα+ CD11c+ cDC by CLOD-LIP. Sirpα and CD11c staining of cells in peripheral blood and thymus at 12 and 24 hours in mice injected with PBS-LIP and CLOD-LIP. B) cellularity C) CD4SP and D) Foxp3+CD4SP cells in 3A9 mice injected with PBS-LIP or CLOD-LIP prior to injection with 50µg HEL (black) or PFS (white). Graphs are from events gated on total lymphocytes and pooled from two experiments: PBS-LIP+PFS (4 mice), PBS-LIP+HEL (6 mice), CLOD-LIP +PFS (4 mice), CLOD-LIP + HEL (7 mice). Statistical significance is indicated on graphs with (*), denoting p<0.05, and (**) denoting p<0.01. Error bars indicate SEM.

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