Variations of this allodepletion strategy have been attempted by other groups, with
differences in the activation antigen used, type of stimulator cell employed, method of
depletion, the in vitro or in vivo readout for depletion and the time frame involved
(Fehse et al. 2000), (Mavroudis et al. 1996), (Harris et al. 1999), (Garderet et al. 1999),
(Mavroudis et al. 1998), (van Dijk et al. 1999), (Datta et al. 1994), (Rencher et al.
CD69 was chosen over CD25 were already discussed in the previous chapter but CD25
alone has been selected by some groups, most commonly with the use of immunotoxins
conjugated with a monoclonal antibody against CD25 (Cavazzana-Calvo et al. 1994),
(Datta et al. 1994), (Mavroudis et al. 1998), (Harris et al. 1999). This were either ricin,
pseudomonas or diphtheria toxin based. The level of specific depletion (7.6+1.4%)
achieved with the pseudomonas based immunotoxin was comparable to the CD69
strategy although retention of 3”^^* party reactivity (64.2+5.0%) was not as well
preserved (Mavroudis et al. 1996). Impressive results were obtained with the ricin
based toxin (6% specific depletion and 92.7% 3*^^ party retention) (Cavazzana-Calvo et
al. 1990) but this was not tested in an HLA-matched setting. The use of immunotoxins
however, results in the inevitable loss of the activated alloreactive cells. Cellular
recovery may be poor and it is difficult to estimate the actual level of depletion
achieved. It also requires the manufacture and the approval of the immunotoxins for
chnical use. The risk of non-specific toxicity may be a potential problem. The removal
of CD25+ alloreactive cells by immunomagnetic separation seemed more attractive in
view of the potential disadvantages of immunotoxins (Garderet et al. 1999), and an
efficient selective depletion of anti-host reactivity was achieved. This however has
again been achieved only in MHC-mismatched pairs and not in the matched setting.
Another potential useful activation marker is 0X40. The association of 0X 40 and
GvHD has been highhghted and in EAE, selective depletion of 0X40+ cells with a
ricin conjugated immunotoxin was successful in amehorating the disease although this
same strategy has not been apphed in the prevention of GvHD. However, restriction of
its expression to CD4+ cells would severely reduce its applicabihty to GvHD
prophylaxis. Other activation markers hke HLA-DR and CD71 have not been used
singly in allodepletion strategies and only in combination with CD25 and CD69.
alloreactive cells and this was done based on both cell size and a combination of
activation markers (CD25, CD69 and HLA-DR). It is however a slow process and
designed for positive rather than depletion selection. Cell recovery was also very poor,
estimated at no more than 10% (Rencher et al. 1996). A combined strategy of removal
of CD25+ and CD69+ cells by immunomagnetic separation was attempted with good
results (75% depletion of the alloreactive response removed) (Fehse et al. 2(X)0).
Though this was only tested in mismatched pairs, its rationale and approach was
complementary to that described in this chapter and would be discussed at greater
length in chapter 8.
In terms of the stimulator cell population, recipient PBMCs have been uniformly used
except in three instances. Recipient kératinocytes were chosen by one group (van Dijk
et al. 1999) with the rationale being that they represented one of the theoretical key
target tissues in GvHD. However, it has been shown that kératinocytes lack expression
of costimulatory molecules important for efficient antigen presentation and hence the
strategy also required the prior transfection of kératinocytes with B7.1 or B7.2. This
meant having to obtain biopsies from patients pre-transplant and transfecting them with
B7.1 or B7.2. The other stimulator cell population used was patient derived B
lymphoblastoid cell lines (BLCL) in view of their high level of MHC antigen
expression (Rencher et al. 1996). The prime concern envisaged with this approach
however remains the issue of safety in using an EBV transformed cell hne for chnical
use. Moreover, it is a labour intensive and time consuming process. Retention of anti-
EBV reactivity, one of the main aims of this strategy may also be lost due to the use of
an EBV transformed cell as stimulator. PHA blasts have been used as stimulators for a
CD25 immunotoxin strategy (Datta et al. 1994) but would seem unsuitable as any
72-96 hours was chosen to be the optimal time for separation and this was in agreement
with other groups using immunomagnetic separation with a combination of activation
markers including CD69. 48 hours was the time point chosen by Fehse for the
combined immunomagnetic separation of CD25 and CD69 but it was acknowledged
from the kinetics of antigen expression that later time points (72 and 96 hours) for
separation might be indicated (Fehse et al. 2000). If the depletion was delayed to 120
hours, the alloantigen specific depletion was found to be noticeably reduced (van Dijk
et al. 1999). With immunotoxins conjugated to anti-CD25, depletion was performed at
earlier time points (48-72 hours). The temporal dynamics of CD25 expression would
suggest that this would not be the optimal time for depletion although it has been
argued that the use of immunotoxins might be able to target very low expressing
CD25+ cells. A 1:1 stimulator/responder (S/R) ratio was uniformly used and it was
shown that with a S/R ratio of 1:2, specific depletion was incomplete while with a S/R
ratio of 2:1, 3"^^ party response was less well preserved (Mavroudis et al. 1996).
This allodepletion strategy has largely been successful in mismatched or haploidentical
pairs but the difficulty remains to translate this work into matched donor-recipient
pairs. This has been reported by 2 other groups-one using kératinocytes as stimulator
cells followed by depletion of the alloreactive lymphocytes with immunomagnetic
separation and the other using PBMCs in a simple MLC with an immunotoxin for
removal (Mavroudis et al. 1998), (van Dijk et al. 1999). The latter method was a little
surprising in that a simple MLC involving matched pairs should result in no
upregulation of CD25. The level of CD25 expression was not documented but the
likeliest explanation remains that perhaps immunotoxins are able to target very low
The cytokine based modified MLC was used by us as the in-vitro test of alloreactivity.
Other assays used have included HTLp and CTLp (Mavroudis et al. 1998), (van Dijk et
al. 1999). All have variously been shown to be predictive for GvHD. A recent study
has highhghted the reliabihty and superiority of the skin explant test over the HTLp
and CTLp assays and hke the cytokine based modified MLC, the skin explant model is
hnked with the crucial role of cytokines in the pathogenesis of GvHD (Dickinson et al.
1998). Perhaps, this assay could be performed in parallel with the cytokine based
modified MLC as further corroboration of our findings.
Preservation of party reactivity has been used as a surrogate marker for the retention
of anti-viral and anti-leukaemic specificity. Specific anti-leukaemic activity has been
measured indirectly with the retention of reactivity to patient PMBC containing
leukaemic blasts (van Dijk et al. 1999), by hmiting dilution assays (LDAs) (Montagna
et al. 1999) and by the conservation of myeloid-specific anti leukaemic reactivity due
to the restricted expression of some mHags when using lymphocytes as stimulators
(Mavroudis et al. 1998). Retention of anti-viral activity against was demonstrated by
showing that the allodepleted cells were able to proliferate against Candida and CMV
antigens. The same group extended this study further by measuring CTLp frequencies
against CMV and EBV infected target cells (Valteau-Couanet et al. 1993), (Montagna
et al. 1999). It remains crucial to ascertain if the allodepleted product contains anti
CMV and EBV-specific lymphocytes and this forms the basis for the results described
in chapter 6.
Attempts have been made to retrovirally insertion a suicide gene into a donor/recipient
cell coculture (Herpes simplex type 1 thymidine kinase gene) together with ganciclovir.
administration of ganciclovir would preferentially target these cells leading to an arrest
of DNA synthesis and cell death. (Bonini et al. 1997), (Mavilio et al. 1994). This
strategy however, requires that all alloreactive cells incorporate the retroviral vector.
Allodepletion with a-CD69 has several advantages over the protocols discussed above:
i) CD69 is upregulated early and on all cells of relevance in GvHD
ii) the MACS columns enable a good median depletion efficiency to be achieved
(80.3%) as well as good cellular recovery. The depleted fraction demonstrated
good cell purity (range 94-99.8%). The superiority in terms of depletion
efficiency of the MACS separation columns over Dynal bead has been
demonstrated and corroborated by another group (van Dijk et al. 1999)
iii) the use of immunomagnetic bead columns is already in chnical use for CD34
selection as a sterile procedure
iv) it is easy, reproducible and the degree of depletion can be reliably quantified
unhke immunotoxin.
v) the alloreactive cells are easily recoverable and can be cryopreserved for ready
immediate access. This would be useful if taken into the chnical arena as DLI if
the patient relapses. This could be of particular value in the treatment of
relapsed acute leukaemias, especially if donor availabihty and recall is a
problem