[Elimination of leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes specifically targeting WT1-derived peptide: an experimental study in vitro].
OBJECTIVE
To investigate the effects of targeting elimination of the leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes (CTLs) specifically against Wilms tumor gene (WT1)-derived peptide.


METHODS
A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A * 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups: Group D (pure T cells) and Group C (IL2 + T cells). The dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A* 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A * 0201 (Group A). The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34+ progenitor cells positively expressing WT1 (3 being HLA-A * 0201(+) and 3 being HLA-A*0201(-)), peripheral blood CD34+ cells from healthy persons (2 being HLA-A * 0201(+) and 1 being HLA-A * 0201(-)), and leukemia cells of the lines of NB4 (HLA-A * 0201(+)/WT1(+)), U937 (HLA-A * 0201(+)/WT1(-)), and K562 (HLA-A * 0201(-)/WT1(+)) were measured by methyl thiazolyl tetrazolium (MTT) chromatometry assay. The colony-forming unit-granulocyte macrophage (CFU-GM) forming capability of the leukemic bone marrow CD34+ progenitor cells and peripheral blood CD34+ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methylcellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading(Group B)and T cells cultured with IL-2 (Group C) were taken as controls.


RESULTS
The CTLs specific for WT1 peptide and restricted by HLA-A * 0201 (Group A) exerted a specific lysis upon the 3 cases with HLA-A * 0201(+)/WT1(+) leukemic bone marrow CD34+ progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% +/- 2.8%, 67.1% +/- 3.2%, 49.4% +/- 3.8% and 55.0% +/- 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201(-)/WT1(+) leukemic bone marrow CD34+ progenitor cells, normal healthy peripheral blood CD34+ cells of the healthy persons, and leukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%). The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01). The relative colony formation rates of WT1-CTLs in Group A upon the 2 cases with HLA-A * 0201(+)/WT1(+) leukemic CD34+ progenitor cells were 17.8% +/- 4.0% and 20.8% +/- 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B (88.9% +/- 3.4% and 91.8% +/- 5.7% respectively, both P < 0.01). There was no significant difference in the relative colony formation rate upon HLA-A * 0201(+)/WT1(-) leukemic bone marrow CD34+ progenitor cells and the HLA-A * 0201(-)/WT1(-) or HLA-A * 0201(+)/WT1(-) normal peripheral blood CD34+ progenitor cells between the CTLs of Groups A and B.


CONCLUSIONS
The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34+ progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34+ progenitor cells. The product of expression of WT1 gene may be used as a target in the leukemic CD34+ cells.
Gu
Wei-ying
WY
Department of Hematology, First People's Hospital of Changzhou, Third Hospital Affiliated to Soochow University, Changzhou 213003, China.
Chen
Zi-xing
ZX
Qiu
Guo-qiang
GQ
Hu
Shao-yan
SY
Jia
Li
L
Wu
Wei
W
He
Jun
J
Cen
Jian-nong
JN
He
Bai
B
D000328:Adult; D018952:Antigens, CD34; D001854:Bone Marrow Cells / Q000276:immunology*; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D016162:Genes, Wilms Tumor; D055014:Granulocyte-Macrophage Progenitor Cells / Q000276:immunology; D015234:HLA-A Antigens / Q000276:immunology; D015789:HLA-A2 Antigen; D006801:Humans; D007938:Leukemia / Q000628:therapy*; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D025721:WT1 Proteins / Q000235:genetics*
D018952:Antigens, CD34; D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D010455:Peptides; D025721:WT1 Proteins