12th Congress of the European Hematology ... - Haematologica

Immunology and gene therapy
0184
EARLY RECOVERY OF THYMUS-DERIVED NAÏVE T CELLS AND OF NK CELLS IN
PEDIATRICS PATIENS AFTER T-CELL DEPLETED HLA-HAPLOIDENTICAL STEM CELL
TRANSPLANTATION FOR THALASSEMIA
A. Isgrò, 1 P. Sodani, 1 M. Marziali, 1 P. Polchi, 1 B. Erer, 1
J. Gaziev, 1 K. Paciaroni, 1 A. Roveda, 1 L. Spitaleri, 1 W. Leti, 2
M. Campagna, 2 F. Aiuti, 2 G. Lucarelli1 1Mediterranean Institute of Hematology, ROMA; 2Dept. of Allergy & Clinical
Immunology, ROMA, Italy
Background. Delayed immune recovery post transplant remains a significant
obstacle and results in increased risk of infections. T cells are
regenerated via 2 pathway, thymus-derived and peripheral expansion,
processes for which IL-7 is critical. Methods. To analyse the mechanisms
involved in immunological reconstitution, we studied six thalassemia
patients after 20 and 60 days post T-cell-depleted HLA-haploidentical
stem cell transplantation. The mean age ranged from 14 to 5 years. As
controls, 6 healthy donors matched by sex and age with the patients
were included. We analysed T cell subsets by flow cytometry. Stromal
cells, obtained from long term culture of bone marrow mononuclear
cells were analysed by immunohystochemistry and the stromal IL-7 production
was analysed by ELISA. Results. Day + 20 post transplant, the
patients had significantly lower CD4 + T cells in comparison to the controls
(1.9±1.4% vs. 47.5±6% respectively), and this reduced number was
mainly observed in CD45RA + CD62L + (naïve phenotype) subset (1.3±2%
in patients vs. 52±12% in controls). A significant decrease of peripheral
CD45RA + CD31 + Th cells (thymic naïve Th cells) (on average 0.5±0.3%
in patients vs. 37±10% in controls) was observed, whereas CD8 + T cells
numbers did not statistically differ between patients and controls
(24.2±33.7% vs. 20±7%). NK cells were among the first lymphocytes to
repopulate the peripheral blood, and up to 70% of these cells were CD56
bright whereas CD56dim CD16 + NK cells were reduced. Day + 60 post
transplant an increase in the percentages of CD4 + T cells, naïve CD4 + cells
and in thymic naïve Th cells were observed (3±1.2%, 2.9±2.1%,
2.7±1%, respectively). CD8 + T cells were also increased (in mean
35±27.5%). Compared with normal subjects, thalassemia patients
showed a significant increase of CD4 + cell activation markers (CD95,
HLA-DR and CCR5) and this was observed after 60 days post transplant,
in parallel with the increase of the CD56 dim CD16 + NK cells especially
in the patients with full engraftment. Stromal cells secreted lower
IL-7 levels (0.3+0.1 pg/mL vs. 0.8+0.1 pg/mL, in controls) and displayed
by immunohistochemistry an altered phenotype (macrophage-like morphology).
Discussions. A significant decrease in total lymphocyte counts
and depletion of CD4 + T cells expressing predominantly the
CD45RA + CD62L + phenotype were observed after 60 days post transplant.
Also the CD4 + CD45RA + CD31 + T cell subset was initially reduced
but an increase has been observed at day + 60 post transplant, suggesting
a thymus involvement in these patients. An IL7/IL7R pathway dysregulation
has been also observed, possibly involving bone marrow stromal
cells. NK cells were among the earliest lymphocytes to repopulate
the peripheral blood, but. CD56 dim CD16 + NK cells were increased after
60 days post transplant, especially in the patients with full engraftment,
suggesting a role of donor NK cells on bone marrow engraftment. We
hypothesize that the recovery of T cell compartment may be due to an
altered production of new T cells starting from the haematopoietic stem
cells under the influence of stromal cytokines production.
0185
VACCINATION WITH DENDRITIC CELLS: CELLULAR IMMUNE MODULATION BY
LEUKOCYTAPHERESIS IN PATIENTS WITH METASTATIC MALIGNANT MELANOMA
M. Hendelmeier, 1 A. Oehring, 1 M. Erdmann, 2 J. Doerrie, 2 N. Schaft, 2
E. Kohler, 3 R. Zimmermann, 1 E. Strasser, 1 R. Eckstein1
1 2 Transfusion Medicine, ERLANGEN; Department of Dermatology, ERLAN-
GEN; 3Center for Molecular Medicine, ERLANGEN, Germany
Background. Monocyte-derived Dendritic Cells (DC) are promising
tools for the cellular immunotherapy of cancer (Banchereau et al, Nature
Rev Immunol 2005; 5:296-306). Monocytes are, however, immune competent
cells and consist of different subpopulations (Gordon et al, Nature
Rev Immunol 2005; 5:953-964) with different physiological roles (Clancy
et al., J Leukoc Biol 2006; 79:757-766). However, no data is available
yet considering the impact of leukocytapheresis on monocyte subpop-
ulations in patients with metastatic malignant melanoma. Aims. Leukocytapheresis
is a stimulus for recruitment of distinct monocyte subpopulations
in patients and the enrichment of monocytes in apheresis products.
The focus is on the main population of CD14 ++ monocytes and the
subpopulations of CD14 + CD16 + monocytes, CD33dimCD16 + monocytes,
and CD33bright monocytes. The impact of the apheresis procedure
on these important monocyte subpopulations was demonstrated.
Methods. 18 patients with metastatic malignant melanoma and 22
healthy blood donors were investigated before and after leukocytapheresis
procedures. Additionally, monocyte apheresis products (MAP) of
patients were compared with the results of healthy blood donors. The
percentage of monocyte subpopulations were investigated by flow
cytometry (FACS Calibur, BD). The impact of the apheresis procedure
was demonstrated by calculation of the recruitment factor (RF). Calculation
formula: RF = (postdonation cell count + cell yield) / predonation
cell count. Results. Monocyte subpopulations showed different enrichment
in patients, in healthy blood donors and in MAP. In patients (n=18)
with metastatic malignant melanoma the CD14 + CD16 + monocytes
increased by 57% (p=0.002) in the postdonation count. Additionally, the
CD33dimCD16+ monocytes increased by 40.3% (p=0.002) whereas the
same cell population in healthy blood donors decreased by 12%
(p=0.079) in blood counts after apheresis procedure. CD14 + CD16 + monocytes
were enriched in MAP by factor 2 (4.99±2.53% vs. 10.14±6.71%,
p