Reduced CTLA-4 protein and messenger RNA expression in umbilical cord blood T lymphocytes.
OBJECTIVE
A favorable incidence and severity of graft-vs-host disease is observed in patients transplanted with banked, unrelated, HLA-mismatched umbilical cord blood (UCB) grafts, while the incidence of malignant relapse remains low. CTLA-4 mediates negative T-cell signaling and may contribute to the development of allogeneic tolerance. In this study, we compared protein and mRNA expression of CTLA-4 in stimulated UCB and adult peripheral blood T cells.


METHODS
T cells were isolated from UCB and adult peripheral blood and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Cells were immunostained and analyzed by flow cytometry for both surface and intracellular expression of CTLA-4 in the presence and absence of cyclosporin A, and kinetics of CTLA-4 expression compared. CTLA-4 mRNA expression was measured using quantitative real-time polymerase chain reaction. NFAT1 protein levels were measured by Western blot analysis.


RESULTS
These studies demonstrate reduced surface and intracellular expression of CTLA-4 in stimulated UCB T cells compared to adult controls. Furthermore, reduced CTLA-4 protein expression in UCB T cells was noted to be in part transcriptionally regulated, as CTLA-4 mRNA levels also were significantly lower. Reduced CLTA-4 expression by UCB T cells followed the kinetics of delayed and reduced expression of the transcription factor NFAT1 by UCB T lymphocytes during primary stimulation. Moreover, cyclosporin A, which is known to modulate NFAT activation, reduced CTLA-4 protein expression in adult and UCB T cells.


CONCLUSIONS
Reduced expression of the key regulatory proteins CTLA-4 and NFAT-1 may contribute to favorable UCB T lymphocyte allogeneic responses.
Department of Pediatrics, University Hospitals Ireland Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA.
Robin E
RE
Miller
John D
JD
Fayen
Shaden F
SF
Mohammad
Kevin
K
Stein
Suzanne
S
Kadereit
Kathleen Daum
KD
Woods
R Michael
RM
Sramkoski
James W
JW
Jacobberger
Dennis
D
Templeton
Susan B
SB
Shurin
Mary J
MJ
Laughlin
D000069594:Abatacept; D000328:Adult; D000375:Aging / Q000276:immunology; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics; D060908:CTLA-4 Antigen; D002455:Cell Division / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects; D006086:Graft vs Host Disease / Q000276:immunology; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects; D006801:Humans; D007108:Immune Tolerance; D018796:Immunoconjugates; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007231:Infant, Newborn; D008213:Lymphocyte Activation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D004268:DNA-Binding Proteins; D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D050778:NFATC Transcription Factors; C494006:NFATC2 protein, human; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D000069594:Abatacept; D016572:Cyclosporine