Figure 1.
Culture and enrichment of tDCs.
Phase bright (A) and phase contrast (B) images of hematopoietic cultures showing non-adherent cells with dendritic morphology. Flow cytometry forward/side scatter profile of cultured cells before and after enrichment (C). Gates indicate tDC population; typical purity after enrichment was 70–80%. (D) Low magnification of Giemsa-Wright stained cytospin of enriched cells. (E) High magnification of Giemsa-Wright stained cytospin of enriched cells.
Figure 2.
tDCs express MHCII and stimulate the MLR more effectively than B cells or macrophages.
(A) tDCs are positive for surface MHCII by flow cytometry (black line histogram: MHCII hyper-immune serum, grey filled histogram: pre-immune serum control), but not IgM (B, black line histogram: anti-IgM, grey filled histogram: secondary antibody alone). (C) When compared to resting peritoneal macrophages (black dotted line histogram) and B cells (black solid line histogram), tDCs (solid grey histogram) express higher levels of MHCII on their surface. (D) tDCs were cultured with CFSE stained spleen responders in a primary allogeneic MLR at tDC to responder ratios from 1∶1 to 1∶8. Numbers represent percent dividing responders. (E) Allogeneic primary MLRs were conducted with tDCs, macrophages, or B cells as stimulators. Percent dividing cells was determined by flow cytometry analysis of CFSE dilution. Data are representative of 3 independent experiments, each using stimulators and responders from multiple individual fish.
Figure 3.
Ultrastructural features of tDCs.
(A) Transmission electron micrographs of representative tDCs (scale bars = 2 µm). (B) High magnification of tDC cytoplasm detailing lamellar structures seen in some cells (arrows). (C) High magnification of tubular mitochondria (arrow), Golgi (arrow head) and smooth ER (D) characteristic of tDCs. Scale bar in B = 0.2 µm, C and D = 0.5 µm.
Figure 4.
(A) Fluorescence of tDCs incubated for 2 hours with 1 µM opsonized fluorescent latex beads (black line) compared to tDCs alone (grey filled histogram). Gate indicates percent phagocytic cells (81.2%). Representative data are shown (mean percent phagocytic tDCs for experiment was 68.6%, n = 5). Experiment was repeated 2 times. (B) Cytospin showing internalized beads (beads indicated by “x”) in a representative tDC, compared to a representative macrophage (C) under the same conditions (too many beads to mark).
Figure 5.
tDCs were stained with CFSE and injected intraperitoneally into recipient fish. Twenty-four hours later, the number of CFSE positive, live events in one million collected events per tissue was determined by flow cytometry. The number of CFSE positive events in all tissues was totaled and events per tissue were divided by this total to arrive at percent of migrating cells in each tissue. Data are from four independent experiments. Error bars indicate SEM.
Figure 6.
RT-PCR was performed for trout homologs of mammalian DC markers of interest. (A) tDCs express mRNA for TLR-3, -5,-9,-20,-22, and-22L. (B) tDCs express mRNA for the B7 costimulatory molecules B7R, B7H1, B7H3, and B7H4. (C) tDCs express mRNA for molecules involved in DC function including: IL12p40, CXCR4, CCR7, MHCII, CD83, and CD209. Results for tDCs from six individual fish are shown. PCRs shown were performed in parallel. No PCR products were detected in no RT controls or no template controls (GPDH no RT controls shown, other data not shown). (D) Quantitative real-time RT-PCR analysis of CD83 and MHCII mRNA expression in tDCs compared to muscle (error bars indicate SD, *p<0.05).
Figure 7.
Activation of tDCs by TLR-ligands.
Phase contrast images show aggregation of tDCs typical of activated leukocytes after 24 hrs incubation with TLR-ligands (A), while tDCs in medium alone remained randomly distributed (B). tDC CD83 mRNA copy numbers, measured by real-time RT-PCR, increase significantly (*p<0.05) compared to media controls at 24 hrs of exposure to TLR-ligands (C), while MHCII mRNA copy numbers are not significantly changed (D). However, after 4 day incubation with TLR-ligands, tDCs upregulate surface expression of MHCII (E), (grey dotted histogram: TLR-ligand treated tDCs, black line histogram: media control tDCS, and grey filled histogram: pre-immune serum control) and by cytospin take on a morphology strikingly similar to mammalian DCs (F).
Figure 8.
Isolation of tDC-like cells from the spleen and culture from peripheral blood mononuclear cells.
(A) Phase contrast images of tDC-like cells isolated from spleen using a mammalian protocol. Isolated cells are initially adherent (2 hr) but become non-adherent overnight, unlike macrophages from peritoneal lavage that remain adherent. (B) Non-adherent cells isolated from spleen express surface MHCII (grey filled histogram: pre-immune serum, black line histogram: anti-MHCII hyper-immune serum). (C) Phase contract image of adherent cells from mononuclear fraction of peripheral blood after 2 hr incubation. (D) Phase contrast of typical culture of adherent peripheral blood cells (20×, day 18). (E) Cytospin of peripheral blood cultures reveal the presence of large cells with long veils and kidney shaped nuclei (40×, day 7).