Molecular strategies for detection and quantitation of clonal cytotoxic T-cell responses in aplastic anemia and myelodysplastic syndrome.
Immune mechanisms are involved in the pathophysiology of aplastic anemia (AA) and myelodysplastic syndrome (MDS). Immune inhibition can result from cytotoxic T cell (CTL) attack against normal hematopoiesis or reflect immune surveillance. We used clonally unique T-cell receptor (TCR) variable beta-chain (VB) CDR3 regions as markers of pathogenic CTL responses and show that while marrow failure syndromes are characterized by polyclonal expansions, overexpanded clones exist in these diseases and can serve as investigative tools. To test the applicability of clonotypic assays, we developed rational molecular methods for the detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 AA, 37 MDS, and 21 paroxysmal nocturnal hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, respectively. In total, 81 immunodominant signature clonotypes were identified. Based on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays for monitoring corresponding clones, including clonotypic Taqman polymerase chain reaction (PCR) and clonotype-specific sequencing. No correlation was found between clonality and disease severity but in patients treated with immunosuppression, truly pathogenic clones were identified based on the decline that paralleled hematologic response. We conclude that immunodominant clonotypes associated with marrow failure may be used to monitor immunosuppressive therapy.
Wlodarski
Marcin W
MW
Experimental Hematology and Hematopoiesis Section, Taussig Cancer Center R/40, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, USA.
Gondek
Lukasz P
LP
Nearman
Zachary P
ZP
Plasilova
Magdalena
M
Kalaycio
Matt
M
Hsi
Eric D
ED
Maciejewski
Jaroslaw P
JP
D000595:Amino Acid Sequence; D000741:Anemia, Aplastic / Q000235:genetics* / Q000276:immunology*; D001483:Base Sequence; D016022:Case-Control Studies; D002999:Clone Cells / Q000276:immunology; D022801:Complementarity Determining Regions; D018076:DNA, Complementary / Q000235:genetics; D019674:Genes, T-Cell Receptor beta; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics; D007125:Immunogenetics; D008969:Molecular Sequence Data; D009190:Myelodysplastic Syndromes / Q000235:genetics* / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D022801:Complementarity Determining Regions; D018076:DNA, Complementary; D016056:Immunodominant Epitopes