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In vivo detection of dendritic cell antigen presentation to CD4(+) T cells.

Ingulli E, Mondino A, Khoruts A, Jenkins MK - J. Exp. Med. (1997)

Bottom Line: DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells.These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes.This interaction results in T cell activation and disappearance of the DC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

ABSTRACT
Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4(+) T cells specific for an OVA peptide-I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

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In vivo clustering of  OVA peptide–MHC-bearing  DC and DO11.10 T cells.  DO11.10 T cells (green) and DC  (red) were purified, dye-labeled  and injected into recipient mice  as described in Materials and  Methods. Draining popliteal  lymph nodes were harvested 4, 8,  24, and 48 h after DC injections.  Tissue was processed and analyzed by confocal microscopy as  described in Materials and Methods. The optical thickness of  each image is 4.5–5 μm. Images  were taken from paracortical regions of lymph nodes of mice injected with (A) OVA peptidepulsed DC or (B) unpulsed DC.  Bar, 100 μm.
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Figure 2: In vivo clustering of OVA peptide–MHC-bearing DC and DO11.10 T cells. DO11.10 T cells (green) and DC (red) were purified, dye-labeled and injected into recipient mice as described in Materials and Methods. Draining popliteal lymph nodes were harvested 4, 8, 24, and 48 h after DC injections. Tissue was processed and analyzed by confocal microscopy as described in Materials and Methods. The optical thickness of each image is 4.5–5 μm. Images were taken from paracortical regions of lymph nodes of mice injected with (A) OVA peptidepulsed DC or (B) unpulsed DC. Bar, 100 μm.

Mentions: Using immunohistochemical detection with the KJ1-26 mAb, we previously showed that DO11.10 T cells were present in the T cell–rich paracortical regions of all lymph nodes within 24 h of intravenous injection (5). Similarly, intravenously injected, CMFDA-labeled DO11.10 T cells were found in the paracortical regions of the lymph nodes (Fig. 1) 24 h after injection, indicating that the dye labeling process did not affect their trafficking ability. In the absence of antigen, the CMFDAlabeled DO11.10 T cells could be detected via their green fluorescence in the paracortical regions for at least 72 h (data not shown). CMTMR-labeled, unpulsed DC were first detected in the draining lymph nodes 8 h after subcutaneous injection, accumulated to a maximal level by 24 h, and declined slightly by 48 h (Figs. 2 B and 3 A). By 24 h, the unpulsed DC were found almost exclusively in the paracortical regions of the lymph nodes (Fig. 1 B). Despite the paracortical colocalization of the labeled T cells and unpulsed DC, few if any interactions between the cells were observed throughout the 48-h observation period (Figs. 1 B, 2 B, and 3 B).


In vivo detection of dendritic cell antigen presentation to CD4(+) T cells.

Ingulli E, Mondino A, Khoruts A, Jenkins MK - J. Exp. Med. (1997)

In vivo clustering of  OVA peptide–MHC-bearing  DC and DO11.10 T cells.  DO11.10 T cells (green) and DC  (red) were purified, dye-labeled  and injected into recipient mice  as described in Materials and  Methods. Draining popliteal  lymph nodes were harvested 4, 8,  24, and 48 h after DC injections.  Tissue was processed and analyzed by confocal microscopy as  described in Materials and Methods. The optical thickness of  each image is 4.5–5 μm. Images  were taken from paracortical regions of lymph nodes of mice injected with (A) OVA peptidepulsed DC or (B) unpulsed DC.  Bar, 100 μm.
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Related In: Results  -  Collection

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Figure 2: In vivo clustering of OVA peptide–MHC-bearing DC and DO11.10 T cells. DO11.10 T cells (green) and DC (red) were purified, dye-labeled and injected into recipient mice as described in Materials and Methods. Draining popliteal lymph nodes were harvested 4, 8, 24, and 48 h after DC injections. Tissue was processed and analyzed by confocal microscopy as described in Materials and Methods. The optical thickness of each image is 4.5–5 μm. Images were taken from paracortical regions of lymph nodes of mice injected with (A) OVA peptidepulsed DC or (B) unpulsed DC. Bar, 100 μm.
Mentions: Using immunohistochemical detection with the KJ1-26 mAb, we previously showed that DO11.10 T cells were present in the T cell–rich paracortical regions of all lymph nodes within 24 h of intravenous injection (5). Similarly, intravenously injected, CMFDA-labeled DO11.10 T cells were found in the paracortical regions of the lymph nodes (Fig. 1) 24 h after injection, indicating that the dye labeling process did not affect their trafficking ability. In the absence of antigen, the CMFDAlabeled DO11.10 T cells could be detected via their green fluorescence in the paracortical regions for at least 72 h (data not shown). CMTMR-labeled, unpulsed DC were first detected in the draining lymph nodes 8 h after subcutaneous injection, accumulated to a maximal level by 24 h, and declined slightly by 48 h (Figs. 2 B and 3 A). By 24 h, the unpulsed DC were found almost exclusively in the paracortical regions of the lymph nodes (Fig. 1 B). Despite the paracortical colocalization of the labeled T cells and unpulsed DC, few if any interactions between the cells were observed throughout the 48-h observation period (Figs. 1 B, 2 B, and 3 B).

Bottom Line: DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells.These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes.This interaction results in T cell activation and disappearance of the DC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

ABSTRACT
Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4(+) T cells specific for an OVA peptide-I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell-rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

Show MeSH
Related in: MedlinePlus