F ig u r e 3.4 C D 3 4 + derived DCs M LR.
D C s ( ■ ) o r m onocytes (O) w ere used as stim ulators and allogeneic M N C s w ere u se d as responders in an M L R . -^H-thymidine incorporation, which quantifies D N A sy n th esis and th u s cell proliferation, w as m e asu red . D C s can be seen to induce a greater d eg re e o f proliferation o f M N C s than m onocytes. T h is e x p e rim en t is representative o f 4 experim ents. E ach data point is th e m ean o f 3 replicates. E rro r b ars represent SEM .
u 4 0 0 0 0
PPD
3 0 0 0 0 - 20000- 10000- 1000 10 100 DC+ no Ag DC+ Ag Mono+ no Ag Mono+ AgStimulator Cell No (xlO^)
12500
KLH
10000- 7 5 0 0 - 5 0 0 0 - 2 5 0 0 - 100 10 DC no Ag DC + Ag Mono no Ag Mono + AgStimulator Cell No (xlO^)
F ig u r e 3.5 C D 34+ derived D C s. C D 4+ T c e ll proliferative response to recall and p rim a ry antigens. G ra p h s sh ow ing the C D 4+ T cell proliferative response, as m easu red b y ^H -thym idine uptake, in resp o n se to antigens presented either by autologtxis C D 34+ derived D C s (♦ ) o r m onocytes K L H w as used as a prim ary antigen and P P D as a recall antigen. D C s w ith o u t an tig en (□ )
and m onocytes w ithout antigen (o) w ere also incubated witli C D 4 + cells as controls. D C s p u lse d w idi p rim a ry o r recall antigens can be seen to induce greater proliferation o f au to lo g o u s C D 4 T cells than c o m p a re d with m onocytes o r D C s not exposed to exogenous antigen, indicating that th e in vitro g en e ra te d D C s are able to stim ulate both naive and m emory T -cells. E ach data p o in t is the m ean o f 3 replicates. E rror bars represent SEM. n=2
noticeably diminished if the cells were pre-incubated with mannan, which inhibits uptake via the mannose receptor (Fig 3.6). MNCs were used as controls.
3.2.7 Generation of DCs from peripheral blood 0014+ monocytes is accompanied by a reduction in cell number.
Peripheral blood monocytes were positively selected from the blood of normal donors on the basis of their expression of CD 14+. Monocytes selected in this way were at least 95% pure by morphology. These cells were cultured in RPMI/10% FCS supplemented with GM-CSF and IL-4, for 7 days. At the end of the culture period there was a
reduction in cell number with only 56.5%±6.3% of the original number of cells remaining (mean±SD; n=10). (Table 3.3).
3.2.8 The majority of cells generated from CD14+ monocytes have the appearance of DCs.
Monocytes which had been cultured for 7 days in the presence of GM-CSF and IL-4 were examined under phase contrast. The cells were non-adherent or loosely adherent, being dislodged by gentle repeated pipetting. The vast majority of cells were large and had numerous cytoplasmic projections characteristic of DCs. Cytospin preparations were made and stained using MGG stain. Again the vast majority of cells had the appearance of DCs, being large cells with a single central nucleus and ample basophilic cytoplasm with numerous fine cytoplasmic projections (Fig 3.7).
3.2.9 The cell surface immunophenotype of monocyte derived DCs
The DCs generated after 7 days of culture were stained with fluorochrome-conjugated antibodies, either directly or indirectly, and analysed by flow cytometry. Over 95% of cells expressed HLA DR and the co-stimulatory molecule CD40. The majority of cells expressed C D la and HLA DQ. The co-stimulatory molecules CD80 and CD86, and the antigens detected by the antibodies CMRF44 and anti-CD83 which are considered to be relatively specific for DCs, were expressed at low to moderate levels. By day 7, less than 10% of cells still expressed CD 14 (Fig 3.8 and Table 3.4).
3.2.10 Monocyte derived DCs are potent stimulators in a MLR.
The alloreactivity of the monocyte derived DCs were compared to that of monocytes from the same donor in a one way MLR. Allogeneic MNCs were used as responder cells. As shown in figure 3.9, DC cultures induced marked proliferation of responder cells over 5 days when compared with autologous monocytes, as determined by ^H- thymidine incorporation. The level of proliferation of MNCs increased as the number of DCs per well increased.
1 8 .0 0 ■ 1 6 .0 0 O 1 4 .0 0 1 2 . 0 0 1 0 . 0 0 ■ 4 . 0 0 2 . 0 0 0 . 0 0 ■ DC □ DC + mannan □ MNC □ MNC + mannan 0 6 0 tim e (m in u te s)
F ig u re 3.6 U ptake o f FITC-Dextran by CD 34+ DCs and MNCs.
M ean cell fluorescence (m et) o f cells at 0 aixi 60 minutes after incubation with
F IT C -dextran in the presence and absence o f mannan is shown. DCs can be seen to have a greater capacity to take up FITC-dextran. This is inhibited to some extent by preincubation w ith m annan, Indicating that some o f the uptake o f FITC-dextran occurs via the m annose receptor. This data is the mean o f 3 experim ents. Error bars indicate the SD.
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