Abstract
Inhibitors of JAK2-kinase (Ruxolitinib, Momelotinib) are already approved or currently investigated in advanced clinical trials for treatment of myeloproliferative neoplasia (MPN). Besides their effect on mutated JAK2-kinase these compounds inhibit wildtype JAK and thereby impair JAK-STAT-signaling, which is an important pathway for proliferation and activation of other cell types such as human T-cells. Accumulating evidence suggests that they may also exert substantial immunosuppressive activity. Very recent reports highlighting hepatitis B reactivation complemented the series of severe infections in ruxolitinib-treated patients among which cryptococcus neoformans pneumonia, toxoplasmosis retinitis, disseminated tuberculosis, and progressive multifocal leukencephalopathy are the most alarming.
We hypothesized that JAK-kinase inhibitors may act as immunosuppressant drugs by impairment of T-cell responses through inhibition of T-cell signaling (JAK-STAT pathway) and that specificity of JAK-kinase inhibition may be of major importance for the degree of T-cell inhibition. Therefore we investigated the effects of pharmacological JAK-kinase inhibition on healthy donor (HD-) and MPN patient T-cells. Selective inhibitors of JAK2-kinase (BSK805) and JAK3-kinase (BQM245) as well as clinically relevant inhibitors of JAK1/2-kinases (Ruxolitinib and Momelotinib) were used for pharmacologic inhibition. The SRC-kinase inhibitor Dasatinib served as a positive control for T-cell inhibition. Knockdown of specific JAK-kinases by RNAi was used to control for target specificity.
In regard to T-cell receptor (TCR)-mediated signaling we investigated bona fide signaling molecules downstream of the TCR by Western Blotting. Besides SRC-kinases like LCK also ZAP70, PLCG1 and the MAPK/ERK pathway have been described to play a pivotal role in T-cell activation. In our data set, selectivity of JAK-kinase inhibition (JAK2, JAK3 or JAK1/2) influenced TCR-signaling in regard to overall tyrosine phosphorylation but also in regard to downstream effectors such as ERK.
As activation and proliferation of primary T-cells is a critical step in immune responses against viral and tumor antigens we aimed to investigate the influence of JAK-kinase inhibition on activation and proliferation of human T-cells. T-cells from healthy donors were stimulated using either PHA 0.5% or CD3/CD28 beads to ensure a more T-cell receptor specific stimulation. CD69 expression was used as a marker for T-cell activation and CFSE staining was applied to assess for T-cell proliferation. Using CD3/CD28 stimulation, CD69 expression was almost abrogated following Dasatinib treatment and proliferation was significantly reduced. Applying relevant doses of specific JAK2 and JAK3 inhibitors to isolated T-cells did neither influence CD69 expression nor T-cell proliferation. These findings are confirmed by RNAi. In contrast, clinically relevant doses of JAK1/2 inhibitors Ruxolitinib and Momelotinib, respectively reduced CD69 expression and T-cell proliferation. Likewise, T-cells derived from MPN patients treated with Ruxolitinib revealed decreased CD69 expression and decreased proliferative capacity upon stimulation, compared to untreated patients or HD-controls.
In order to investigate T-cell function, we assessed for allo-reactivity in a mixed lymphocyte culture. Human pan-T-cells were co-cultured with allogeneic antigen presenting cells. T-cell reactivity – as measured by 3H-thymidine incorporation – was significantly impaired by Ruxolitinib and Momelotinib. Specific inhibition of JAK2 or JAK3 kinase, however, did not affect T-cell reactivity. These effects could be confirmed using T-cells derived from Ruxolitinib-treated MPN patients. Investigation of leukemia- and virus-antigen-specific T-cell responses are currently under way to gain deeper insight regarding this clinically relevant scenario.
Taken together, specificity of JAK-kinase inhibition influences the inhibitory potential on T-cell function. JAK1 kinase seems to play an important role in T-cell activation, as unspecific inhibitors of JAK1 & JAK2 Kinase inhibit T-cell function while selective inactivation of JAK2 kinase leaves T-cell function almost unaffected. Heterogeneity in T-cell function of Ruxolitinib-treated patients is an important finding that deserves detailed investigation.
Disclosures
Heidel: Novartis: Consultancy.
T cell co-stimulatory receptor CD28 is a primary target for PD-1–mediated inhibition
