3,1 Introduction:
Fisure 3.6 Temporal expression o f CD69 in CD4/8 cells after alloseneic stimulation o
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96 tim e in hours 120 144 192 d iffe re n c e in % C D 4 + C D 6 9 + activation d iffe re n c e in % C D 8 + C D 6 9 + activationFigure 3.7 Temporal expression o f CD69 in CD56+ cells after alloseneic stimulation
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tim e in hoursd iffe re n c e in % C D 5 6 + C D 6 9 + activ atio n
control cultures: responder cells alone or with autologous PK H labelled stim ulators. For CD 56+ cells, the % CD 69 expression was defined as the proportion o f C D 56+C D 69+ cells divided by the total proportion o f C D 56+ cells. The sam e was done for CD4+ and C D 8+ cells. All data are expressed as m ean j^s.d.
3.2.5 PKH26 does not alter the allostimulatorx capacity o f the M LC
To exclude the possibility that PKH26 labelling might directly affect CD69 expression,
a time course experiment was performed directly comparing control responder cells
alone or with PKH 26 labelled autologous stimulator cells. This showed no non
specific increase in CD69 expression using the PKH26 labelled cells as compared to
the control unlabelled responder population (data not shown).
It was also necessary to demonstrate that PKH26 labelhng did not reduce the
allostimulatory capacity of the allogeneic PBMCs. To do this, CD69 expression in a
one way MLC with similar donor-stimulator pairs were compared using irradiated
versus PKH26 labelled stimulators. At 72 hours and beyond, both irradiated and PKH
labelled stimulators could be easily resolved from the responders and for the earlier
time points, stimulator PBMCs were irradiated 48 hours earUer. No significant
difference was found in the allostimulatory capacity between the two. In subsequent
experiments using irradiated stimulators, the temporal dynamics of CD69 expression
was also found to be broadly similar. This showed that PKH26 labelhng did not affect
CD69 activation in an MLC. It also confirmed that effectiveness of PKH labelhng in
3 .2.6 Tem poral dynam ics o f activation m arker C D 25 in a one wav M L C
Initial experiments performed with PKH labelled stimulators showed no CD25
expression above basehne control up to 48 hours. A time course study was therefore
done using responders and MHC mismatched irradiated stimulators. The activation
profiles of CD25, CD69, 0X 40 and HLA-DR were compared using the panel of
antibodies described in material and methods. Figure 3.8 shows the expression of
CD25 in various lymphocyte subsets after alloantigen stimulation. In contrast to CD69,
there was a detectable but small upregulation of CD25 (1.3%) above basehne control
only at 72 hours. There was a further small rise at 96 hours (mean CD25 expression
above basehne of 3.0%). At later time points however (120 hours and beyond), there
was a sharp rise in CD25 expression to a peak of 43.2% indicating sustained level of
expression. This was associated with increasing cell size and a skewing of the MLC to
the prohferating alloreactive cells. CD25 expression was restricted only to the CD3+ T
cell subset with no expression on the CD3- lymphocytes including NK cells. Within
the CD3+ T cells, this was largely a CD4+ response with only a small proportion of
CD8+ cells expressing CD25 and only at very late time points (144 hours and beyond).
CD25 was expressed at sufficient density to allow for clear distinction between the
positive (CD25 expressing) and negative population on a dot-plot.
When cells were double stained for CD25-FITC and CD69-PE simultaneously, there
were no double positive activated ceUs seen for the first 72 hours of the MLC. Only
CD69 was expressed on activated cells. Significant numbers of CD25+CD69+ cells
were seen (1.5% above basehne) only at 96 hours which corresponded with the
temporal dynamics of CD25 expression. The proportion of double positive activated
expression of CD25 at this time point. This also indicated the stabihty of CD69
expression in that it was still expressed past peak levels. If only the “blastoid” or
proliferating cells were analysed, at 72 hours, 80% of the cells were CD69+ but only
5% CD25+; at 96 hours, the proportion of CD25+ cells had increased to 54% while the
CD69 expression remained stable. At 120 hours onwards, 70% of the cells were
CD25+CD69+ and by 168 hours, nearly all proliferating cells expressed CD25 while
Figure 3.8 CD25 expression in lymphocytes with alloantisen stimulation 50n 45- ë 4 w- 30- o 25- y 154 10- 5- 0 La a — Qj
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24 48 72 96 time in hours 120 144 192 difference in %CT)25+ activation difference in % CD4+25+ activation difference in % CD8+25+ activation difference in % CD3+25+ activation difference in % CD3-25+ activationThe percentage change o f activation antigen CD 25 above baseline autologous control (y-axis) was analysed against tim e in hours from the start o f the one way M LC (x-axis). The percentage change was calculated at each tim e point using the follow ing equation: (% o f lym phocytes expressing the relevant activation m arker in the M LC - % of lym phocytes expressing the sam e m arker in the autologous control culture). The percentages w ere calculated at each tim e point using quadrant analysis o f dot-plots. CD25 expression was also analysed for the follow ing subsets: CD 3+, CD 4+, C D 8+ and CD3- lym phocytes. O nly lym phocytes which fell inside the lym phocyte gate were considered in the analysis. All bar colum ns in the figure are expressed as m eans + s.d.
3,2 .7 Tem poral dynam ics o f activation m arker 0 X 4 0 in a one wav M L C
As anti-OX40 PE was only available as a PE-conjugate, labelling of stimulator cells
with PKH26 in this case was not the appropriate choice. Activation involves the
upregulation of only a small proportion of alloreactive cells and as PKH26 fluoresces in
the high FL2 region, the possibility of a significant error in results could arise if any of
the PKH26 labelled stimulators were erroneously included as an activated OX40PE4-
cell. PKH2 labelhng of stimulator PBMCs which fluoresces in the ELI region was
attempted but the resolution of FITC-labelled responder PBMCs with PKH2 labelled
cells was poor. This was because unhke PKH26, there was a gradient of labelhng with
PKH2 and this overlapped with FITC positive cells. Increasing the concentration of the
dye could not rectify this problem. F18 labelhng, which also fluoresces in the FLl
region was attempted with no improvement in resolution. Irradiated 2-day-old
stimulator cells were then used to allow for analysis of responder cells at 24 and 48
hour time points and freshly irradiated cells for time points thereafter.
Figure 3.9 shows the temporal dynamics of 0X 40 expression with allostimulation. The
earhest time point when 0X 40 expression was first detected was at 72 hours (1.8%),
similar to CD25 and in contrast to CD69 where expression was seen as early as 24
hours. Thereafter, there was a steady increase, reaching a peak at 120 to 144 hours
(mean 4.4% above basehne). The decrease in levels thereafter signified that the
expression of 0X 40 might not be sustained. 0X 40 was only seen on CD3+ T cells and
not at all on CD3- lymphocytes including NK cells. Within the CD3+ T cell subset,
nearly all cells expressing 0X 40 were CD44- cells, with minimal expression on CD8+
cells (fig 3.9). 0X 40 was expressed at sufficient density to allow for clear distinction
Fi2ure 3.9 0 X 4 0 expression in T cells with alloantisen stimulation in an M LC