Abstract Book 2010 - CIMT Annual Meeting

036 Wagner | Immune monitoring
GPI-anchor negative T cells with impaired effector function persist
after Alemtuzumab mediated T-cell depletion
Eva Maria Wagner 1 , Aline Naomi Lay 1 , Caroline Goetze 1 , Diana Wolff 1 , Julia Hemmerling 1 ,
Matthias Theobald 1 , Wolfgang Herr 1 , Ralf Georg Meyer 1
1 Department of Hematology/Oncology at the University Medical Center of the Johannes Gutenberg-University
Mainz, Germany
The monoclonal anti-CD52 antibody Alemtuzumab
is frequently used for T-cell depletion (TCD) in the
context of allogeneic hematopoietic stem cell transplantation
(HSCT). We have previously demonstrated
that substantial proportions of reconstituting T
cells remained CD52 negative for years, especially
in patients who did not receive donor lymphocyte
infusions. CD52 is a glycosylphosphatidylinositol
(GPI)-anchored molecule of unknown function. By
flow-cytometry, we demonstrated that the lack of
CD52-expression was associated with decreased
expression of further GPI-anchored molecules like
CD55 and CD59. We therefore analyzed CD52 negative
T cells of 20 patients after HSCT with a fluorescent
aerolysin staining (FLAER) that directly labels
the GPI-anchor and found that the loss of CD52 and
further GPI-anchored proteins in T cells was due to
the loss of the GPI-anchors themselves. In contrast,
CD52 positive T cells of the same patients showed
no reduced FLAIR-signal and normal expression
of CD55 and CD59. Patients who underwent Alemtuzumab-mediated
TCD prior to allogeneic HSCT
suffer from long-lasting immunologic dysfunction
even when peripheral T cell counts have reconstituted
to normal range. Hence, we compared GPI-anchor
positive and negative T cells for their response
to viral and allogeneic stimuli. When FACS-sorted
CD52 negative and positive T cell populations were
expanded in vitro (IL2, OKT3, feeder-cells), we observed
that GPI-anchor expression remained absent
in the CD52 negative T-cell cultures. In contrast, the
CD52 positive T-cell cultures showed constant expression
of GPI-anchors for more than 16 weeks. In
addition, the CD52 expression had no influence on
the growth-kinetics following antigen-independent
stimulation with IL2 and OKT3. However, CD52 positive
T cells showed enhanced proliferation after specific
stimulation with CMV-pp65 peptides. In IFN-γ
ELISPOT assays using CMV-peptide (pp65 and IE-1)
loaded autologous dendritic cells as stimulators,
specific spot production was only detected among
the GPI-anchor positive T cells. To investigate on
T-cell function ex vivo, we performed IFN-γ Secretion-assays
with T cells directly isolated from patients.
CD52 negative T cells again showed a reduced
IFN-γ-secretion after stimulation with either autologous
DCs loaded with the CMV-peptides or with
allogeneic DCs. Nonetheless, T cells with CMV-pp65
specific T cell receptors were present in the CD52
positive as well as the CD52 negative subpopulations,
as proven by tetramer-staining. Despite the
functional differences, the percentages of naïve and
memory T cells did not differ between both populations.
In summary, we demonstrated that after Alemtuzumab-based
TCD, substantial proportions of
reconstituting donor T cells lost the expression of
GPI-anchors. GPI-anchor negative T cells showed
reduced proliferative capacity as well as reduced
IFN-γ secretion in response to CMV or allogeneic
stimulation. Our data suggest that a loss of GPI-anchored
molecules might contribute to an impaired
function of reconstituting donor T cells after Alemtuzumab-mediated
TCD. We will further investigate
on the impact of different GPI-anchored molecules
on effector-functions of different T-cell subsets.
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