Suppression of primary T-cell responses and induction of alloantigen-specific hyporesponsiveness in vitro by the Janus kinase inhibitor tyrphostin AG490.
BACKGROUND
Tyrphostin AG490 has recently been shown to block interleukin (IL)-2 receptor gamma-chain-associated Janus kinase 3. Here, we analyzed the effect of AG490 on T-cell alloresponses in vitro.


METHODS
For the evaluation of T-cell activation, DNA synthesis, surface marker expression, cytokine secretion, intracellular calcium mobilization, early protein tyrosine phosphorylation, and apoptosis were measured.


RESULTS
AG490 effectively inhibited T-cell proliferation in human mixed lymphocyte culture (MLC) even when added 4 days after culture initiation. Inhibition of IL-2-dependent proliferation in T-cell blasts and the incapability of IL-2 or IL-15 to restore proliferation in AG490-treated MLC suggests interference with cytokine receptor signaling. T-cell receptor-triggered early protein tyrosine phosphorylation, calcium mobilization, up-regulation of CD69, and initial CD25 expression were not affected. Interestingly, AG490 substantially inhibited production of IL-2 and interferon-gamma in T cells stimulated with alloantigen or via CD3 and CD28. In CD28-independent activation models (e.g., stimulation with phorbol myristate acetate plus ionomycin), however, cytokine secretion was not inhibited. Pretreatment of primary MLC with AG490 resulted in substantial down-regulation of secondary responses to cells from the original donor as opposed to third-party cells or phytohemagglutinin. Unresponsiveness was induced also in T cells stimulated with CD3 monoclonal antibody. Induction of apoptosis in polyclonally activated T cells and the incapability of IL-2 to reverse specific hyporesponsiveness, suggest programmed cell death as an important mechanism underlying antigen-specific down-regulation of alloresponses.


CONCLUSIONS
We demonstrate that AG490 blocks different manifestations of T-cell activation. This and its ability to induce alloantigen-specific hyporesponsiveness point to a potential use for interfering with alloreactivities in vivo.
Säemann
M D
MD
Institute of Immunology and Department of Internal Medicine III, University of Vienna, Austria.
Böhmig
G A
GA
Osterreicher
C H
CH
Staffler
G
G
Diakos
C
C
Krieger
P M
PM
Hörl
W H
WH
Stockinger
H
H
Zlabinger
G J
GJ
D017252:CD3 Complex / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000276:immunology; D015536:Down-Regulation; D006801:Humans; D053616:Janus Kinase 3; D007959:Lymphocyte Culture Test, Mixed; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects / Q000502:physiology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D020032:Tyrphostins / Q000494:pharmacology*
D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D011505:Protein-Tyrosine Kinases; C507933:JAK3 protein, human; D053616:Janus Kinase 3; D002118:Calcium