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Identification of a novel antigen cross-presenting cell type in spleen.

Tan JK, Quah BJ, Griffiths KL, Periasamy P, Hey YY, O'Neill HC - J. Cell. Mol. Med. (2010)

Bottom Line: L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c(lo) CD11b(hi) MHC-II(-) CD8α(-) cells, distinct by high accessibility and endocytic capacity for blood-borne antigen.However, they have weak ability to stimulate CD4(+) T cells in antigen-specific responses.Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.

View Article: PubMed Central - PubMed

Affiliation: Research School of Biology, The Australian National University, Canberra, ACT, Australia.

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Antigen-presenting capacity of L-DC. CD8α+ cDC, myeloid cells (mono/mac) and L-DC subsets were sorted from C57BL/6J spleen based on gating protocols described in Figure 4. LTC-DC were collected as non-adherent cells from LTC established from spleens of B6.SJL mice. f-DC were enriched from spleens of C57BL/6J and CBA/H mice using CD11c+ microbead positive selection (MACS). (A) To measure in vitro cross-presenting ability, L-DC and CD8α+ cDC were pulsed with OVA (10 μg/ml) and co-cultured with purified CFSE-labelled CD8+ T cells (105) from OT-I mice ±LPS (1 μg/ml). Proliferation of cells was measured by dilution of CFSE after 4 days for calculation of percentage divided cells. (B) Isolated L-DC and CD8α+ cDC were pulsed with OVA (as in A), and 104 cells injected i.v. into B6.SJL (CD45.1) mice previously injected with 105 CFSE-labelled OT-I lymphocytes. Spleens were harvested at 34 days following rechallenge of mice at 25 days with OVA (10 μg, i.v.), and assessed flow cytometrically for CFSE and total number of OT-I CD8+ T cells in spleen. (C) L-DC and LTC-DC were compared with APC subsets isolated from spleen for capacity to induce a MLR. Responder T cells were prepared from CBA/H mice by depletion of B cells, myeloid cells and DC using magnetic beads, labelled with CFSE, and co-cultured with APC, using T cells only as a control. Responding T cells were analysed flow cytometrically at 4 days following antibody staining to gate live (PI−), CD11c−, CD4+ or CD8+ populations. Number of divided cells reflects cells showing a reduction in CFSE intensity.
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fig06: Antigen-presenting capacity of L-DC. CD8α+ cDC, myeloid cells (mono/mac) and L-DC subsets were sorted from C57BL/6J spleen based on gating protocols described in Figure 4. LTC-DC were collected as non-adherent cells from LTC established from spleens of B6.SJL mice. f-DC were enriched from spleens of C57BL/6J and CBA/H mice using CD11c+ microbead positive selection (MACS). (A) To measure in vitro cross-presenting ability, L-DC and CD8α+ cDC were pulsed with OVA (10 μg/ml) and co-cultured with purified CFSE-labelled CD8+ T cells (105) from OT-I mice ±LPS (1 μg/ml). Proliferation of cells was measured by dilution of CFSE after 4 days for calculation of percentage divided cells. (B) Isolated L-DC and CD8α+ cDC were pulsed with OVA (as in A), and 104 cells injected i.v. into B6.SJL (CD45.1) mice previously injected with 105 CFSE-labelled OT-I lymphocytes. Spleens were harvested at 34 days following rechallenge of mice at 25 days with OVA (10 μg, i.v.), and assessed flow cytometrically for CFSE and total number of OT-I CD8+ T cells in spleen. (C) L-DC and LTC-DC were compared with APC subsets isolated from spleen for capacity to induce a MLR. Responder T cells were prepared from CBA/H mice by depletion of B cells, myeloid cells and DC using magnetic beads, labelled with CFSE, and co-cultured with APC, using T cells only as a control. Responding T cells were analysed flow cytometrically at 4 days following antibody staining to gate live (PI−), CD11c−, CD4+ or CD8+ populations. Number of divided cells reflects cells showing a reduction in CFSE intensity.

Mentions: CD8α+ cDC are highly cross-presenting cells both in vitro [32] and in vivo [2, 17]. We therefore compared L-DC with CD8α+ cDC for cross-presenting ability in vitro. APC subsets were isolated and pulsed with OVA or HEL and co-cultured with purified OT-I CD8+ T cells in the presence and absence of LPS as an activator. L-DC were strong activators of OT-I CD8+ T cells but only after LPS activation. The cross-presenting capacity of CD8α+ cDC was very strong, and increased after activation with LPS (Fig. 6A). The main phenotypic effect of LPS activation on L-DC after culture in vitro maps to up-regulation of CD86, which is not expressed on freshly isolated cells (Fig 5; all data not shown).


Identification of a novel antigen cross-presenting cell type in spleen.

Tan JK, Quah BJ, Griffiths KL, Periasamy P, Hey YY, O'Neill HC - J. Cell. Mol. Med. (2010)

Antigen-presenting capacity of L-DC. CD8α+ cDC, myeloid cells (mono/mac) and L-DC subsets were sorted from C57BL/6J spleen based on gating protocols described in Figure 4. LTC-DC were collected as non-adherent cells from LTC established from spleens of B6.SJL mice. f-DC were enriched from spleens of C57BL/6J and CBA/H mice using CD11c+ microbead positive selection (MACS). (A) To measure in vitro cross-presenting ability, L-DC and CD8α+ cDC were pulsed with OVA (10 μg/ml) and co-cultured with purified CFSE-labelled CD8+ T cells (105) from OT-I mice ±LPS (1 μg/ml). Proliferation of cells was measured by dilution of CFSE after 4 days for calculation of percentage divided cells. (B) Isolated L-DC and CD8α+ cDC were pulsed with OVA (as in A), and 104 cells injected i.v. into B6.SJL (CD45.1) mice previously injected with 105 CFSE-labelled OT-I lymphocytes. Spleens were harvested at 34 days following rechallenge of mice at 25 days with OVA (10 μg, i.v.), and assessed flow cytometrically for CFSE and total number of OT-I CD8+ T cells in spleen. (C) L-DC and LTC-DC were compared with APC subsets isolated from spleen for capacity to induce a MLR. Responder T cells were prepared from CBA/H mice by depletion of B cells, myeloid cells and DC using magnetic beads, labelled with CFSE, and co-cultured with APC, using T cells only as a control. Responding T cells were analysed flow cytometrically at 4 days following antibody staining to gate live (PI−), CD11c−, CD4+ or CD8+ populations. Number of divided cells reflects cells showing a reduction in CFSE intensity.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822631&req=5

fig06: Antigen-presenting capacity of L-DC. CD8α+ cDC, myeloid cells (mono/mac) and L-DC subsets were sorted from C57BL/6J spleen based on gating protocols described in Figure 4. LTC-DC were collected as non-adherent cells from LTC established from spleens of B6.SJL mice. f-DC were enriched from spleens of C57BL/6J and CBA/H mice using CD11c+ microbead positive selection (MACS). (A) To measure in vitro cross-presenting ability, L-DC and CD8α+ cDC were pulsed with OVA (10 μg/ml) and co-cultured with purified CFSE-labelled CD8+ T cells (105) from OT-I mice ±LPS (1 μg/ml). Proliferation of cells was measured by dilution of CFSE after 4 days for calculation of percentage divided cells. (B) Isolated L-DC and CD8α+ cDC were pulsed with OVA (as in A), and 104 cells injected i.v. into B6.SJL (CD45.1) mice previously injected with 105 CFSE-labelled OT-I lymphocytes. Spleens were harvested at 34 days following rechallenge of mice at 25 days with OVA (10 μg, i.v.), and assessed flow cytometrically for CFSE and total number of OT-I CD8+ T cells in spleen. (C) L-DC and LTC-DC were compared with APC subsets isolated from spleen for capacity to induce a MLR. Responder T cells were prepared from CBA/H mice by depletion of B cells, myeloid cells and DC using magnetic beads, labelled with CFSE, and co-cultured with APC, using T cells only as a control. Responding T cells were analysed flow cytometrically at 4 days following antibody staining to gate live (PI−), CD11c−, CD4+ or CD8+ populations. Number of divided cells reflects cells showing a reduction in CFSE intensity.
Mentions: CD8α+ cDC are highly cross-presenting cells both in vitro [32] and in vivo [2, 17]. We therefore compared L-DC with CD8α+ cDC for cross-presenting ability in vitro. APC subsets were isolated and pulsed with OVA or HEL and co-cultured with purified OT-I CD8+ T cells in the presence and absence of LPS as an activator. L-DC were strong activators of OT-I CD8+ T cells but only after LPS activation. The cross-presenting capacity of CD8α+ cDC was very strong, and increased after activation with LPS (Fig. 6A). The main phenotypic effect of LPS activation on L-DC after culture in vitro maps to up-regulation of CD86, which is not expressed on freshly isolated cells (Fig 5; all data not shown).

Bottom Line: L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c(lo) CD11b(hi) MHC-II(-) CD8α(-) cells, distinct by high accessibility and endocytic capacity for blood-borne antigen.However, they have weak ability to stimulate CD4(+) T cells in antigen-specific responses.Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.

View Article: PubMed Central - PubMed

Affiliation: Research School of Biology, The Australian National University, Canberra, ACT, Australia.

Show MeSH
Related in: MedlinePlus