2.5 COMMUNITY MANAGEMENT
2.5.1 MODELO DE COMMUNITY MANAGEMENT
The results presented in this chapter describes the effect of alloantigen stimulation on
the expression profile of various activation markers on the responding lymphocyte cell
surface, namely CD69, CD25, 0X 40 and HLA-DR. The parameters analysed which
would be of particular relevance to a selective depletion strategy and to the
pathophysiology of GvHD were the time course of upregulation, peak levels and
density of expression as well as the pattern of distribution among the various
lymphocyte subsets.
All the activation antigens studied demonstrated selectivity of response in that
expression remained low without an alloantigen stimulus while in the presence of
allogeneic PBMCs in an MLC, there was a consistent upregulation of cell surface
activation markers. This specificity was further confirmed when it was shown that the
allogeneic response was not affected by either the PCS used in the culture or by the
process of PKH26 labelhng. All this suggested that it was possible to identify
alloreactive responder cells by virtue of their expression of activation antigens. Of the
four activation antigens studied, CD69 was the first to be expressed following
alloantigen stimulation at 24 hours, followed by 0X 40 (72 hours) and CD25 (72 hours)
while HLA-DR was the last activation antigen to be expressed. The time taken to peak
levels of expression was also in that order, with CD69 peaking at 96-120 hours and
HLA-DR at 192 hours. If alloreactive cells causing GvHD could be identified using
these activation markers, the advantage of choosing CD69 over the others would be the
rapidity of its expression and the abihty to detect and isolate maximum numbers of
alloreactive cells (peak level of expression) in the shortest time interval. This was an
clinical GvHD prevention strategy. Any procedure that involves the minimal time for
manipulation or ex-vivo culture would be advantageous in terms of sterility and
logistics involved. Moreover, it was noted that beyond 96 hours, there was a
progressive skewing of the responder cell population as the alloreactive cells begin to
divide and numbers increase disproportionately. In contrast, the non-alloreactive cell
population which contains useful mature cells possibly involved in anti-viral and anti
leukaemia activity would most hkely start to undergo apoptosis the longer they remain
in ex-vivo culture without receiving any productive stimulus. This skewing of the
responder cell population was evidenced by the fact that at 144 hours, over 40% of the
cells were activated (CD25+ and HLA-DR+) and within the increasing “blastoid” gate,
nearly all were activated. Therefore, it would be important if these alloreactive cells
could be identified before considerable skewing of the responder population occurs at
120 hours and beyond. Only CD69 and 0X 40 were expressed at appreciable levels by
96 hours.
All four activation markers were expressed in sufficient density for clear separation and
identification using flow cytometry. As such, this was not a factor in deciding the
optimal marker to use although it has been reported that CD69 expression exhibits a
higher fluorescence intensity compared to CD25 (Cebrian et al. 1988). With regards to
the spectrum of cellular distribution, only CD69 was expressed on CD4+, CD8+ T cells
and NK cells which have all been implicated in the pathogenesis of GvHD. CD25 was
also expressed on both CD4+ and CD8+ T cells but there was no evidence of
expression on NK cells in the MLC system that was used. In contrast, 0X 40 was
found to have a restricted cellular distribution, confined mainly to CD4-k T cells alone
and minimal expression on CD8+ cells which is consistent with other reports in the
but the problem remains that HLA-DR is constitutively expressed on B cells,
monocytes and dendritic cells. Any selective depletion strategy based on the removal
of HLA-DR expressing cells would also remove those cells in addition to activated T
cells from the graft. This might have profoundly detrimental effects on post-transplant
immune reconstitution.
For all those reasons mentioned above, CD69 was selected as the most suitable marker
for the allodepletion strategy. Although the lymphocyte response in terms of CD69
expression to potent stimuh like PMA and PHA have been well studied (Craston et al.
1997), (Caruso et al. 1997), there have been only a few reports regarding the
upregulation of CD69 in response to an alloantigen stimulus. Potent stimuli like PHA
induce a non-specific response from the majority (60-90%) of T cells while the data
presented in this chapter show that with alloantigens, only a minority of T cells express
CD69. This small proportion of cells in the MLC (%) that activated in response to the
alloantigen was in keeping with previous estimates of the frequency of alloreactive T
cells (l-5%)(Detours and Perelson, 1999), (Sherman and Chattopadhyay 1993). The
dynamics of the CD69 alloantigen response was also markedly different from the
mitogen responses of both T and NK cells where expression of CD69 was seen as early
as 1 hour after stimulation and peaked 4-24 hours later. In all cases CD69 expression
in response to alloantigen stimulation was delayed with upregulation first seen only at
24 hours. The results of the early time points (24-72 hours) were corroborated by one
group (Fehse et al. 2000) while another study of CD69 expression in response to
alloantigen stimulation by irradiated cells detected expression above baseline but the
earliest time point studied was 60 hours post initiation of the MLC and the response
peaked at 108-156 h with 21% of the cells were CD69+, comparable to the time point
different dynamics of CD69 expression with a peak response observed at 48 hours.
However PBMC sonicates were used rather than whole stimulator cells and this could
possibly alter the time frame of alloreactive recognition and activation (Leiva et al.
1997). Pagüeroni used monocytes as stimulators (1:1 responderistimulator ratio) but
examined only the CD4 response (Paglieroni et al. 1999). A response was first seen at
12 hours, a peak expression at 24h with 2-21% of CD4+CD3+ cells CD69+, and
tapering off at 96h. On the other hand, one report has suggested that the use of CD69
as a method of assessing lymphocyte activation may only be restricted to potent stimuh
with no increase seen in response to alloantigen (Simms & Elhs 1996). However, the
same group failed to detect a CD69 response to tetanus toxoid in contrast to the
findings of at least two others groups (Caruso et al. 1997), (Mardiney et al. 1996). It
may be that in some culture systems failure to detect a CD69 response to specific
stimuh is due to high levels of background cell activation, presumably in response to
cytokines within the foetal calf serum. It is possible that such background activation
may screen the low level alloantigen-specific activation and explain the negative
findings. The experimental systems described in this chapter ensured that the batch of
PCS used did not affect CD69 expression non-specifically.
Another advantage in using CD69 was its stabihty of expression. Although
upregulated early after allostimulation, sustained expression was observed at least up to
144 hours. Double staining showing simultaneous expression of CD25 and CD69 at
144 hours confirmed the finding that CD69 continues to be stably expressed on the cell
surface to overlap with the later expression of CD25.
The results noted that a higher proportion of NK cells responded to the alloantigen
mismatches between the responder-stimulator pair at the appropriate HLA-C and HLA-
B loci involved in NK cell regulation (Colonna & Samaridis 1995). This observation
supports the concept that NK cells are involved in graft rejection and graft-versus-host
disease after allogeneic bone marrow transplantation (Murphy et al. 1993).
This chapter has also described a novel method of estabhshing a one-way MLC to
circumvent the issue of responders and stimulators falling into the same lymphoid gate
and making the enumeration of alloreactive cells difficult. This method of using
PKH26 to label stimulator PBMCs is especially useful when examining early
lymphocyte responses. This problem was tackled in a different way by (Leiva et al.
1997) who used PBMC sonicates rather than whole cells. The problem here was that it
was found that only freshly used sonicates were able to stimulate an allogeneic reaction
and this capacity was lost if cells were cryopreserved. This would therefore not be
apphcable in a chnical allodepletion strategy as recipient and donor samples may not
necessarily be obtained at the same time.
The basehne expression of CD69 in autologous cultures was 2.18% and this was
supported by the findings of other groups (0.8+0.4%) (Cebrian et al. 1988), (Pagheroni
et al. 1999). As CD69 is expressed at low levels on resting lymphocytes, the
percentage of cells that were induced to express CD69 after an alloreactive stimulus
was therefore used a measure of the strength of the response. The other parameter that
may be important is the density of expression as measured by fluorescence intensity.
From the responder-stimulator pairs used, there appeared to be no appreciable
difference in fluorescence intensity from one pair to another but clear distinction could
be seen between CD69 positive and negative cells. In normal donors, the density of
et al. 1995) and would suggest that the use of percentage positive values was a vaUd
index of activation with samples from normal donors.
Standard in vitro methods for assessing T cell activation have typically measured either
the proliferative responses of PBMC cultures to various provocative stimuli employing
tritiated thymidine incorporation or the secretion of specific cytokines. However, these
bulk assay methods suffer the drawback of being lengthy assays and in addition, they
do not provide information about functional responses of individual lymphocyte
subsets. The development of intracellular cytokine staining has allowed the
visualisation of individual cytokine secreting cells. The detection of cell surface
activation antigens is another.
It has been demonstrated that the expression of CD69 on multi-parametric flow
cytometry at 4 hours to a potent stimulus like the comitogenic monoclonal antibodies
CD2/CD2R mirrored closely the dose-response patterns observed with lymphocyte
proliferation as measured by thymidine incorporation (Maino, Suni, & Ruitenberg
1995). Mardiney has also found agreement between percentage of cells expressing
CD69 and proliferation (Mardiney et al. 1996), as did Lamb LS (Lamb, Jr. et al. 1997)
while others report (Caruso et al. 1997) overall agreement between the two in
distinguishing a positive from a negative response but no actual correlation between
percentages and the amount of ^H-thymidine incorporation. In addition, it has been
(Paglieroni et al. 1999) found that not only did CD69 expression compare favourably
with thymidine uptake but also with intracellular cytokine production to an alloantigen
stimulus. Multi-parameter flow cytometry employing FITC-labelled antibodies to
specific Vp T cell receptor antigens found that CD69 expression in response to SEE
(Kappler et al. 1989). This suggested that CD69 expression on activated T cell subsets
also demonstrate specificity at the level of the T cell receptor. The induction of CD69
also strictly correlated with the extent of CD3/TCR cross-linking (Testi et al. 1988).
All these findings imply that CD69 expression is antigen-specific and part of the
integral set of events beginning with TCR engagement and leading to proliferation and
cytokine secretion: for a cell to proliferate, it needs to be activated first (Testi et al.
1989b), (Lanier et al. 1988). The converse however may not hold true in that not all
activated cells would finally proceed to proliferation. PMA alone for example induces
all cells to express CD69 but does not stimulate significant DNA synthesis in the
absence of calcium ionophore. Moreover, if the amount of stimulus was lowered,
CD69 upregulation still occurred but in the absence of prohferation (Caruso et al.
1997). Nonetheless, within the first hours of activation, a number of committed
differentiation pathways consequent to T cell activation become evident (cytokine
expression, apoptosis, anergy) before DNA synthesis and cell division occur (Germain
& Stefanova 1999). Factors that influence this T cell activation response as measured
by CD69 expression include the type of stimulus, accessory cell function, avidity of the
TCR and the amount of costimulatory and adhesion molecules present (Maino et al.
1995).
CD69 has been used as an informative marker in various disease states. Expression of
CD69 has been used to assess the anti CD3 induced proliferative response and predict
functionality of the lymphocytes in HIV infected patients (Nielsen et al. 1998), (Prince
& Lape-Nixon 1997). Eosinophils isolated from the bronchoalveolar lavage of asthma
patients showed abnormally high CD69 cell surface expression compared to normal
allograft rejections selectively express CD69 (Santamaria et al. 1992). CD69 is also
expressed by CD8+ infiltrating cells in the livers of patients with chronic active
hepatitis (Garcia-Monzon et al. 1990). It has been used as a marker to monitor
autoimmune disease hke systemic lupus erythematosus activity (Su et al. 1997) and
rheumatoid arthritis (Isomaki et al. 1997). Other investigators have also used it to
assess T cell function post bone marrow transplantation (Lamb, Jr. et al. 1997).
Therefore, the identification of cell surface structures like CD69, minimally expressed
on resting PBMCs but broadly upregulated on activated lymphocytes following
stimulation would enhance the potential for detection of low frequency responses Hke