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1.
Figure 6

Figure 6. Overview of changes in DC sensitivity. From: LPS Activated Dendritic cells: "Exhausted" or alert and waiting? DCs: "Exhausted", alerted or waiting?.

Resting DCs (residing in tissues) are responsive to one set of signals, such as PAMPs, DAMPs, and other alarm signals (represented here with LPS and LPS + IFNg). They are then thought to change to an “exhausted” state in which they no longer respond to external signals. However, these cells are not “exhausted”, but instead are simply responsive to a different set of signals, such as T cell-derived products and other environmental signals. Depending on their activation state, and on the signals they receive, the DCs produce different amounts of IL-12p75 (and other cytokines).

Kaveh Abdi, et al. J Immunol. ;188(12):5981-5989.
2.
Figure 2

Figure 2. CD40L and IL-4 play an important role in IL-12p75 production by DCs. From: LPS Activated Dendritic cells: "Exhausted" or alert and waiting? DCs: "Exhausted", alerted or waiting?.

BM-DCs from B10.A-Rag2−/− mice were either resting, or pre-activated with 100ng/ml LPS for 27hr then washed and co-cultured at 1×105 cells/well with 5×105 cell/well NIH-3T3 mock transfected, NIH-3T3 CD40L expressing cells or antigen-activated 5C.C7 T cells plus 0.1μM MCC for 48hr in a 48 well plate. CSN was tested for presence of IL-12p75 heterodimer using specific ELISA representative of three independent experiments.

Kaveh Abdi, et al. J Immunol. ;188(12):5981-5989.
3.
Figure 4

Figure 4. Inflammatory cytokine production is not extinguished in LPS pre-activated DCs. From: LPS Activated Dendritic cells: "Exhausted" or alert and waiting? DCs: "Exhausted", alerted or waiting?.

(A) BM-DCs from B10.A-Rag2−/− mice were either at resting state or pre-activated with 100ng/ml LPS for 26hr (“exhausted”) or pre-activated with 100ng/ml LPS plus 100ng/ml rIFN-g for 26hr (“Hammered”) and then washed before being re-stimulated at 2×104 cells/well in a 96 well U-bottom plate with 1×105/well antigen-activated 5C.C7 T cells (TH0), AE7 (TH1 clone) or D10 (TH2 clone) plus 0.1μM MCC peptide or 1μM Conalbumin for 48hr. (B) Same as (Fig. 2) CSN were tested for the presence of various cytokines using multiplex cytokine array. (A) Sum of three independent experiments except TH0 (B) sum of two independent experiments except IL-1α.

Kaveh Abdi, et al. J Immunol. ;188(12):5981-5989.
4.
Figure 5

Figure 5. Production of various T cell cytokines in the presence of resting, “exhausted” and “hammered” DCs. From: LPS Activated Dendritic cells: "Exhausted" or alert and waiting? DCs: "Exhausted", alerted or waiting?.

BM-DCs from B10.A-Rag2−/− mice were either resting, or pre-activated with 100ng/ml LPS for 26hr (“exhausted”) or pre-activated with 100ng/ml LPS plus 100ng/ml rIFNg for 26hr (“Hammered”) and then washed before being re-stimulated at 2×104 cells/well in a 96 well U-bottom plate in the final volume of 200μl with 1×105/well antigen-activated 5C.C7 T cells (TH0), AE7 (TH1 clone) or D10 (TH2 clone) plus 0.1μM MCC peptide or 1μM Conalbumin respectively for 48h. Various T cell cytokines were measured using multiplex cytokine array. These data are expressed as the mean ± SD compilation of three independent experiments.

Kaveh Abdi, et al. J Immunol. ;188(12):5981-5989.
5.
Figure 1

Figure 1. LPS pre-activated DCs are not “exhausted” but make IL-12p75 in the presence of antigen-activated T cells. From: LPS Activated Dendritic cells: "Exhausted" or alert and waiting? DCs: "Exhausted", alerted or waiting?.

(A) BM-DCs from B10.A-Rag2−/− mice were either, resting or pre-activated with 100ng/ml LPS for 27hr, then washed and cultured at 2×104 cells/well in a 96 well U-bottom plate. These cells were re-stimulated with 100ng/ml LPS in the presence or absence of 100ng/ml of rIFNg for 48hr. CSN was tested for the presence of IL-12p75 with specific ELISA. (B) Same as (A) except “exhausted” BM-DCs were pre-activated with LPS for 24hr prior to co-culture with naïve or antigen-activated T cells in the presence of 0.1μM MCC peptide. Each dot represents an independent experiment (activated T cells compilation of 44 experiments; all statistical significant was assessed by Student’s T-test **p <0.002 ***p <0.0001). (C) Same as (A) except one group of BM-DCs (“Hammered”) were pre-activated with 100ng/ml LPS plus 100ng/ml rIFNg for 27hr and then washed before being re-stimulated with LPS, LPS plus IFNg or co-cultured with 1×105/well antigen-activated 5C.C7 T cells plus 0.1μM MCC peptide. (D) Same as (C) except BM-DCs were pre-activated for 25hr. CSN was analyzed for the presence of various cytokines using Searchlight protein arrays. These data are expressed as the mean ± SD compilation of two independent experiments.

Kaveh Abdi, et al. J Immunol. ;188(12):5981-5989.
6.
Figure 3

Figure 3. TH0 and TH1 cells induce IL-12p75 production but TH2 cells do not. From: LPS Activated Dendritic cells: "Exhausted" or alert and waiting? DCs: "Exhausted", alerted or waiting?.

(A) BM-DCs from B10.A-Rag2−/− mice were pre-activated with 100ng/ml LPS for 26hr, then washed and co-cultured at 2×104 cells/well with 5×105 cell/well antigen-activated 5C.C7 T cells (TH0), AE7 (TH1 clone) or D10 (TH2 clone) in the presence of 0.1μM MCC for 5C.C7 and AE7 or 100μM Conalbumin for D10 for 48hr in 96 well U-bottom plates. CSN were tested for the presence of IL-12p75 heterodimer using specific ELISA. (B) Same as (A) except naïve 5C.C7 T cells were cultured with 0.4μM MCC peptide under TH1, TH2 or TH17 skewing conditions. These skewed T cells were sorted for co-culture with “exhausted” BM-DCs. (C) Antigen-activated 5C.C7 T cells were co-cultured with “exhausted” BM-DCs plus 0.1μM MCC in the presence of titrated CSN obtained from APC free D10 TH2 T cell clone that had been stimulated with plate bound anti-CD3 and soluble anti-CD28 (stim) for 48hrs in a 24 well plate. After 48hr CSN were collected and tested for the presence of IL-12p75 with specific ELISA. (D) Same conditions as (C) except some wells received CSN from un-stimulated D10, or from anti-CD3-stimulated D10, or the stimulated CSN, from which IL-10 had been specifically depleted by incubation with protein-G beads coupled with Rat anti-IL-10 monoclonal antibody, or the isotype control. After 48hr, CSN were tested for the presence of IL-12p75 using specific ELISA. (E) Same conditions as (C) except rIL-10 was added to the co-culture assay rather than D10 SN. After 48hr CSN were collected and tested for the presence of IL-12p75 heterodimer using specific ELISA. These data are expressed as the mean ± SD of triplicates representative of three independent experiments.

Kaveh Abdi, et al. J Immunol. ;188(12):5981-5989.

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