Techniques o f T cell depletion:

A number of techniques have been used in achieving T cell depletion. They can be

achieved either by destroying the cells or physically removing them from the graft. In

both cases, the procedure may or may not involve the use of T cell directed monoclonal

antibodies. The most common approaches include:

1) soybean lectin agglutination (SBA) and sheep E rosette form ation (Reisner et

al. 1981). SEA binds and agglutinates cells bearing N-acetyl-D-galactosamine

which is expressed on all mature blood cells, including T cells but not by

pluripotent haematopoietic progenitor cells. A 1.26 ±_0.34 log depletion of T cells

is achieved with this step alone (Keman et al. 1986). Another 1 log T cell depletion

is achieved if combined with E-rosetting in which the CD2 antigen present on T

cells is bound to sheep red blood cells and the complex removed by density

gradient. Besides E rosetting, SEA has also been used in conjunction with panning

on a plastic surface coated with antibodies to CD5 and CD8 (Gajewski et al. 2000)

2) counterflow élutriation (Almici et al 1992), (de Witte et al 1986). This method

separates cells on the basis on their size and density with the use of a spinning

chamber under centrifugal force.

3) Monoclonal antibodies (mAh) used alone or with complement. This rests on the

abihty of cell bound mAb to fix and activate complement.

i) OKT3 (Prentice et al. 1982). This is an anti-CD3 mAh

ii) cocktail of monoclonal anti-T-cell antibodies including anti-CD6, anti-CD7 and

anti-CD8 (Prentice et al 1984), (Mitsuyasu et al 1986).

iii) C am path antibodies (Hale et al. 1990). This is a series of rat mAh directed against

a glycoprotein CDw52 highly expressed on lymphocytes and activates the human

complement cascade. It is reactive to virtually all T and B cells, macrophages,

monocytes, lymphoid leukaemic cells and some NK cells but not to myeloid

leukaemias or stem cells (Hale et al. 1985). Campath-IM (IgM monoclonal) has been

used in combination with donor serum complement to purge T cells from HLA-

matched grafts and was found to be effective in reducing GvHD but graft failure

remained significant (Waldmann et al. 1984). This led to the use of Campath-1G

(IgGzb) administered both in vivo to patients and in-vitro in the graft (Hale et al. 1994),

(Hale et al 1998). Recently, a humanised form, Campath-IH was introduced (Hale et

al. 2000)

4) mAh bound to immunotoxins such as ricin conjugated anti-CD5 (Filipovich et

al. 1990)

5) T cell subset depletion: T10B9. This is an IgM mAh which targets the a p

heterodimer of the TCR and spares the yô T cells which may be involved in a GvL

6) O ther specific T cell antibodies such as CD6 depletion (Soiffer et al. 1997). The

anti-CD6 mAb recognises an antigen that is present on mature T cells but not B,

NK or myeloid precursors. NK cell precursors are therefore spared.

7) Immunomagnetic T cell depletion. This is currently widely used in CD34

selection technology. This method physically separates T cells from the graft,

thereby avoiding the addition of potentially toxic agents and release of cellular

contents from dying cells (de Wynter et al. 1999), (Urbano-Ispizua et al. 1998).

Commonly used immunomagnetic separation techniques include the Dynal beads

which are superparamagnetic, 2.6wm diameter polystyrene beads and the magnetic

cell sorting (MACS) nanoparticles (50nm diameter). When a biotinylated anti

CD34/avidin colunui and an anti-CD34 immunomagnetic bead method was

compared with Campath and complement, the latter achieved a depletion of 2.16

log and a 56% recovery of CD34+ cells while positive selection resulted in 3.12 log

depletion with the avidin colunm and 4.04 log depletion for the bead method and

recovery of CD34+ cells at 27% and 36% respectively (Dreger et al. 1995).

The SBA-erythrocyte rosetting procedure is long and cumbersome and the use of

animal serum and cells may be an issue with regulatory agencies. Counterflow

élutriation requires experienced operators and may be associated with the loss of small

CD34+ stem cells (Noga et al. 1994). A review of results with Campath revealed low

incidence of GvHD but graft failure remains a problem (Hale et al 1994) although this

has been resolved by the additional use of in vivo Campath (Hale et al 1998).

In pan T cell depletion, it has been estimated that the final graft should contain a T cell

dose of <1X10^ T cells/kg recipient body weight to prevent GvHD in matched sibhng

recipients (Verdonck et al. 1994). It however results in a higher incidence of mixed

donor-recipient chimerism after BMT. So far, successful use of T cell depletion has

required the establishment of an appropriate balance between the residual immune

response of the recipient, which can be achieved by enhanced conditioning, and the

adjustment of the T cell content of the donor bone marrow (Lowdell et al. 1998),

(Patterson et al. 1986).

Retrospective analyses of T cell depletion have suggested that certain techniques were

associated with relapse more than others. In particular, physical methods of T cell

depletion and use of narrow spectrum antibodies to deplete the marrow of T cells

reduce the risk of relapse both in matched sibhng and unrelated transplants (Marmont

et al 1991), (Wagner et al. 1990). In a small series using Campath-IM for T cell

depleted matched sibhng allografts for AML in 1®^ remission combined with single

fraction TBI, no severe acute GvHD was seen and the relapse rate was only 14%

(Lowdell and Craston 1997). Thus, TCD BMT after appropriate conditioning may

obviate the issue of graft rejection and prevent GvHD without complete loss of GvL

but the problem of poor immune reconstitution remains.

Specific T cell subset depletion such as CD8+ depletion has been performed on

matched sibhng transplants (Nimer et al 1994) and demonstrated a reduction in GvHD

without an increase in leukaemia relapse. This was corroborated by another group

where the incidence of grade II GvHD was reduced to 22% from 58% using

unmanipulated marrow with a low in cidence of leukaemia relapse although these were

mainly in CML patients (Champhn et al. 1991), (Champhn et al. 1990). CD8 depletion

may also lead to improved CD4 immune reconstitution with concomitant reduction in