Introduction
Non-Hodgkin lymphoma, like follicular lymphoma (FL) and small lymphocytic lymphoma/chronic lymphatic leukemia (SLL/CLL), are hematological malignancies characterized by uncontrolled proliferation of clonal B cells, which leads to painless lymphadenopathy in most cases. Any organ may be affected, which is reflected by the broad variety of clinical presentations of these diseases [
1]. Treatment options for Non-Hodgkin lymphomas have evolved in the past decades from classical chemotherapy to immunotherapy as well as targeted therapy. Among targeted therapies, PI3Kδ has been identified as a potent target for B cell malignancies. PI3Kδ is a tyrosine kinase predominantly expressed in leukocytes. It is located in the cytoplasm and activates the AKT/mTOR pathway [
2]. Idelalisib, a specific small molecule inhibiting PI3Kδ, showed remarkable clinical results for the treatment of FL as well as CLL [
3,
4]. However, infection rates, for example of cytomegalovirus (CMV) and pneumocystis jirocevii (PJP) in patients treated with Idelalisib in combination with classical immune-chemotherapy, were significantly increased [
5]. Impaired T cell-mediated immunity due to direct effects on T cell migration and cytokine production has already been described as a side effect of Idelalisib treatment [
6].
T cell-mediated immunity and the induction of a potent immune response are not only T cell-dependent, but also require professional antigen-presenting cells (APCs). Dendritic cells (DCs) presenting a specific antigen and expressing appropriate co-stimulatory molecules are the most potent APCs. They can orchestrate immune responses of naïve CD8+ T cells and subsequently induce cytotoxic T cell responses [
7].
In this study, we investigated the effect of Idelalisib on DC function, which is a prerequisite for appropriate T cell activation, differentiation and proliferation. Our study may highlight how PI3Kδ inhibition not only diminishes T cell function directly, but also affects antigen-specific T cell responses through attenuated DC function.
Methods
Samples
Human monocytes were isolated from buffy coats from voluntary blood donors at the University Hospital Bonn.
Idelalisib was purchased from Selleckchem. Cells were cultured in RPMI 1640 containing glutamax-I, supplemented with 10% inactivated fetal calf serum (RP10 medium) and 1% penicillin/streptomycin (Invitrogen). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich.
Generation of DCs
Plastic adherence of peripheral blood allowed the generation of human moDCs, as previously described [
8]. These adherent cells were differentiated in RP10 medium. GM-CSF (100 ng/ml; Leukine, Liquid Sargramostim) and IL-4 (20 ng/ml; R&D Systems) were supplemented from the beginning every other day.
Immunostaining
Fluorescence-labeled, monoclonal antibodies commercially available from BD Biosciences, DakoDiagnostika, Immunotech, R&D Systems and eBioscience were used for staining of generated DCs.
Determination of cytokine production
Cytokine secretion was analyzed using the eBioscience™ ProcartaPlex Human Th1 /Th2 Zytokin-Panel (11-plex) (Thermofisher) according to the manufacturer’s instructions.
Mixed lymphocyte reactions
1 × 105 allogeneic peripheral blood mononuclear cells were co-cultured with pretreated and irradiated stimulator moDCs. On day 5, after a 16 h pulse with [3H]-thymidine (18.5 kBq/well; GE Healthcare) Tritium-labeled thymidine incorporation was measured.
Detection of apoptosis
Apoptosis in DCs was detected by live-dead staining using the propidium iodide or 7-aminoactinomycin D–annexin V staining kit from eBioscience.
Polyacrylamide gel electrophoresis and western blotting
Western blotting was performed as described before [
9]. Briefly, whole cell lysates were generated, and protein concentration was measured using a bicinchoninic acid assay (Pierce, Perbio Science, Bonn, Germany). Whole cell lysates (20 µg) were separated on a polyacrylamide gel and transferred onto a nitrocellulose membrane. Monoclonal antibodies by Santa Cruz Biotechnology Inc. (Santa Cruz, USA) were used. An enhanced chemiluminescence kit was used to detect protein bands (GE Healthcare).
Statistical analysis
All experiments were performed at least 3 times, with representative experiments shown. Statistical significance was calculated with one-way analysis of variance (ANOVA) and Dunnett’s using the Prism 8.4.3 software (Graphpad Software).
Discussion
In the past decade, the treatment of Non-Hodgkin B cell lymphoma, such as FL and CLL, has evolved due to the discovery of novel drugs. Among small molecules and targeted therapies, Idelalisib, an inhibitor of PI3Kδ, was a promising new candidate.
Nevertheless, pneumonitis, elevation of transaminases, colitis and diarrhea, as well as life-threatening infections, are serious side effects [
3,
5] and have limited the treatment. Moreover, it has been shown that T cell-mediated immunity is dampened by Idelalisib treatment [
6], and infectious complications are frequent [
5]. Therefore, cytomegalovirus (CMV) monitoring as well as pneumocystis jirovecii pneumonia (PJP) prophylaxis are now mandatory. These infectious complications have led to a decreased and very cautious use of Idelalisib, and its replacement, by other new compounds in many cases in clinical routine [
5]. However, the exact mechanisms mediating the immunosuppressive potential of Idelalisib have not been elucidated in detail yet.
Our report highlights that not only T cells and malignant B cells are targeted by PI3Kδ inhibition but also DC differentiation and function are deeply modified by Idelalisib exposure in vitro. Differentiation of monocytes into moDCs using GM-CSF and IL-4 was markedly impaired by Idelalisib, which modulated proper DC differentiation and phenotype. For example, expression of CD83, which identifies mature DCs and is the most potent co-stimulatory molecule in the induction of allogeneic T cell proliferation, was impaired upon LPS exposure and Idelalisib treatment [
10]. A decrease of CD83 expression due to Idelalisib co-culture may therefore emphasize the immature phenotype of these cells as well as their inability to induce T cell proliferation. As shown previously by a siRNA approach targeting human DCs in vitro [
11], the downregulation of CD83 alone is sufficient for impaired T cell responses, even if expression of other co-stimulatory molecules is unaffected. The impaired DC function is further emphasized by diminished IL-12, IL-13 and TNFα secretion after LPS challenge. IL-13 production by DCs is important to maintain cytokine production in T helper 2 (Th2) cells [
12]. IL-4, which is unchanged by Idelalisib treatment in mature moDCs, induces Th2 differentiation [
13]. IL-13, in contrast, orchestrates survival and functionality of Th2 cells [
12,
14]. Th2-type cytokines are increased during CMV infection [
15] and proper cytokine production by Th2 cells, which is maintained by IL-13 derived from DCs, is necessary for CMV clearance.
IL-12 is a central cytokine in the induction of T cell responses [
16], and adequate release of IL-12 is required to control primary CMV infection in a CD4 + T cell-dependent manner [
17]. Aside from lymphopenia, the impaired release of IL-12 and IL-13 by DCs may thus contribute to the increased susceptibility of Idelalisib-treated patients to CMV infection. Additionally, we were able to show that direct inhibition of the NFκB pathway may be one-way how Idelalisib impairs DC function and phenotype. In line with our data, previous studies could underline the importance of the NFκB pathway in DC development and IL-12 production [
18].
Another serious complication during Idelalisib treatment is PJP infection. It has been shown previously that TNFα is required for clearance of pneumocystis jirovecii [
19] and reduced TNFα levels, as shown here, may be why Idelalisib treatment increases the risk of fatal PJP infection. However, our results are limited because we only investigated the effect of Idelalisib on DC function and not on macrophages, which are a biologically significant source for TNFα.
Last, the inability of Idelalisib pretreated DCs to induce robust immune responses is stressed by the fact that DCs exposed to Idelalisib improperly stimulate allogeneic T cell proliferation, which is another prerequisite to induce a robust immune response after an infectious challenge. DCs activate naïve T cells via 3 signals: Antigen presentation (signal 1), co-stimulation (signal 2) and cytokine production (signal 3) [
20]. Although we did not investigate antigen presentation, we could unveil modulation of co-stimulation and cytokine production by Idelalisib, which is further supported by our results regarding the induction of allogeneic T cell proliferation.
Our results may help to understand the restricted use of Idelalisib due to fatal infectious complications. Idelalisib modulates the expression of at least one co-stimulatory molecule and, more importantly, diminishes cytokine production by DCs in vitro. These cytokines are essential for proper T cell differentiation and polarization. Clinical infection with CMV and PCP may therefore not only be caused by defective T cell responses, but also by insufficient T cell polarization due to diminished cytokine release. Although our data were generated in vitro, clinical observations implicate relevance for our findings in vivo.
In contrast, suppression of T cells and DCs may open new therapeutic venues for PI3Kδ inhibition. For example, in myeloproliferative neoplasms, which are driven by chronic inflammation [
21], a clinical trial combining PI3Kδ inhibition and standard treatment with the JAK inhibitor Ruxolitinib showed promising interim results in patients with myelofibrosis (ClinicalTrials.gov Identifier: NCT02718300). These promising results are supposed to be caused by direct effects of PI3K inhibition on malignant cells [
22], but additional effects as shown in this work have to be investigated as well.
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