GIFT4 fusokine converts leukemic B cells into immune helper cells
© Deng et al. 2016
Received: 12 December 2015
Accepted: 12 April 2016
Published: 27 April 2016
Chronic lymphocytic leukemia (CLL) remains incurable with standard therapy, and is characterized by excessive expansion of monoclonal abnormal mature B cells and more regulatory immune properties of T cell compartment. Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL.
We generated a fusion cytokine (fusokine) arising from the combination of human GM-CSF and IL-4 (named GIFT4). Primary CLL cells were treated with GIFT4 or GM-CSG and IL-4 in vitro. GIFT4-triggered STAT5 signaling in CLL cells was examined by Western blot. The phenotype and secretome of GIFT4-treated CLL cells (GIFT4-CLL cells), and the immune stimulatory function of GIFT4-CLL cells on autologous T cells were analyzed by flow cytometry and luminex assay.
GIFT4-CLL up-regulated the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54. GIFT4-CLL cells secreted IL-1β, IL-6, ICAM-1 and substantial IL-2 relative to unstimulated CLL cells. GIFT4 treatment led to JAK1, JAK2 and JAK3-mediated hyper-phosphorylation of STAT5 in primary CLL cells, which is essential for GIFT4-triggered conversion of CLL cells. GIFT4-CLL cells directly propelled the expansion of autologous IFN-γ-producing CD314+ cytotoxic T cells in vitro, and that these could lyse autologous CLL cells. Furthermore, administration of GIFT4 protein promoted the expansion of human T cells in NOD-scid IL2Rγnull immune deficient mice adoptively pre-transferred with peripheral blood mononuclear cells from subjects with CLL.
GIFT4 has potent capability to converts primary CLL cells into APC-like immune helper cells that initiate a T cell driven anti-CLL immune response.
Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, and it is characterized by excessive expansion of monoclonal abnormal mature B cells in peripheral blood, lymph node, spleen and bone marrow , and remains incurable with standard therapy [2, 3]. T cells in patients with CLL have more regulatory immune response and less immune synapse interaction with antigen-presenting cells (APC) . Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL. Emerging immunotherapies such as chimeric antigen receptor-modified T cells (CART)  and anti-CD20 monoclonal antibodies have shown improved outcomes for CLL management through direct attack on CLL B cells or activating complement membrane attack complex to lyse CLL B cells. However, B cell aplasia, decreased number of plasma cells, hypogammaglobulinemia and life-threatening cytokine release syndrome remain a challenge for CART therapy [6, 7]. Augmented T cell activation and immunity by CD40 ligation  and immune checkpoint blockade , and developing CLL vaccines [9, 10] are under clinical investigation, which provide alternative options for CLL immunotherapy.
We have developed a novel fusion cytokine (fusokine) named GIFT4 that is derived from GM-CSF and IL-4 , built upon the successful development of the GM-CSF and interleukin transgene platforms [12, 13]. GIFT4 protein has potent immune-stimulatory activity on B cells, and induces B cell proliferation through co-clustering of GM-CSF receptor (GM-CSFR) and IL-4 receptor (IL-4R) on the cell surface. GIFT4 treatment further programs naïve B cells into helper cells, which further license anti-tumor T cell response. Considering that CLL B cells express both IL-4R  and GM-CSFR , we tested whether GIFT4 could also have stimulatory effect on CLL cells, and reprogram the leukemic B cells into immune helper cells. Here we show that human GIFT4 stimulation converts primary CLL B cells into APC-like cells with up-regulated expression of co-stimulatory molecules CD40, CD80 and CD86 on their surface. GIFT4-converted CLL B cells (GIFT4-CLL cells) secreted IL-1β, IL-6, ICAM-1 and substantial IL-2, and primed autologous T cells from patients into IFN-γ-producing CD314+ CLL-killer cells.
Characteristics of CLL subjects
CLL subject number
CD5+CD19+ CLL cells
in PBMC (%)
The GIFT4 chimeric transgene was cloned from human GM-CSF and IL-4 cDNA (Invivogen), and the fusion protein was produced in bio-engineered 293T cells as previously described . GIFT4 protein was concentrated and quantified by ELISA kit with anti-human GM-CSF antibodies (eBiosciences, San Diego, CA, USA).
PBMC were isolated from peripheral blood of subjects with CLL using lymphocyte isolation solution (Mediatech). Primary CD5+CD19+ CLL cells were sorted from PBMC on a Becton–Dickinson FACS Aria Cell Sorter. T cells in PBMC were purified with T cell enrichment kits (StemCell). PBMC, primary CLL cells; T cells were cultured in complete RPMI-1640 medium (Corning, Manassas, VA, USA) for 5 days in presence of GIFT4 or recombinant GM-CSF and IL-4 control cytokines (2 ng/ml) (PeproTech). Alternatively, the treated CLL cells were washed with fresh RPMI-1640 medium, and cultured for additional 48 h. Cell culture supernatants were then collected and subjected to luminex assay with human 51plex cytokine polystyrene bead kit (Affymetrix, Santa Clara, CA, USA) in the Human Immunology Monitoring Center at Stanford University. For proliferation assay, PBMC or purified T cells from subjects with CLL were labeled with CFSE dye (Invitrogen, Eugene, OR, USA) following the manufacturer’s instruction, and cultured in complete RPMI 1640 medium for 5 days in presence of GIFT4 or GM-CSF and IL-4 (2 ng/ml). Autologous T cells were co-cultured with GIFT4-CLL cells or control cytokine-stimulated CLL cells (1:1 ratio) for 4 days. T cells were then purified with human T cell positive selection kit (StemCell) and re-cultured (105/ml) in fresh RPMI-1640 medium for additional 48 h. The T cell culture supernatants were subjected to luminex assay.
PBMC from subjects with CLL were stimulated with GIFT4 protein or GM-CSF and IL-4 (2 ng/ml) for 5 days. The cells were harvested and stained with APC-conjugated anti-human CD19 and PE-conjugated anti-human CD5 antibodies; or APC-conjugated anti-human CD3, then subjected to flow cytometry (FACS) on a BD FACSCanto II flow cytometer. Phenotype of GIFT4-CLL cells was profiled by FACS with a panel of B cell antibodies including anti-CD80 and CD86 (BD, San Diego, CA, USA). For analysis of cell–cell interaction between GIFT4-CLL cells and T cells, CFSE-labeled autologous T cells were co-cultured with GIFT4-CLL cells pre-treated with anti-human CD80 or CD86 neutralizing antibodies or isotype control (1 μg/ml) (BioLegend) for 5 days. T cell division was determined by FACS. For intracellular staining of IFN-γ, Granzyme B, and perforin, T cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution, followed by staining with PE-conjugated anti-human IFN-γ, granzyme B, perforin antibodies (BD). Alternatively, circulating human T cells in the peripheral blood of NSG immune deficient mice adoptively transferred with PBMC from CLL patients were profiled and counted by FACS, and analyzed with FlowJo 9.1 software.
The culture supernatants of GIFT4-treated primary CLL cells or control CLL cells were harvested, and the secretome of GIFT4-CLL cells was analyzed by luminex assay with human 51plex cytokine polystyrene bead kit as described .
ELISA and western blot
IL-2 and IL-6 production by CLL cells was quantified with human IL-2 and IL-6 ELISA kit (eBiosciences, San Diego, CA, USA). CLL cells stimulated with human GIFT4 protein, GM-CSF and/or IL-4 (2 ng/ml) cytokines for 20 min in presence or absence of JAK inhibitors  were harvested, and lysed with protein lysate buffer supplemented with protease and phosphatase inhibitors as described . STAT5 phosphorylation in the treated CLL cells was examined by Western blot with anti-pSTAT5 (Tyr694, D47E7) and anti-STAT5 antibodies (Cell Signaling, Boston, MA, USA).
Cell apoptosis assay
GIFT4-CLL primed cytotoxic T cells (105/ml) were co-cultured with primary CLL cells (105/ml) in presence or absence of concanamycin (100 nM) (Sigma) for 24 h in a 96-well plate. The cells were collected and stained with APC-conjugated anti-human CD19 antibodies and Annexin V, then subjected to FACS analysis. Apoptotic cells were gated on Annexin V as previously described .
CLL xenograft in NSG mice
PBMC from subjects with CLL (108 cells/mouse) were adoptively transferred into NOD-scid IL2Rγnull NSG mice  by intravenous injection. The mice were then treated with human GIFT4 protein (20 ng/mouse/day) or control cytokines for 6 days. On day 7, 100 μl of peripheral blood collected from each mouse were stained with anti-human CD3 antibody for 30 min, followed by addition of red blood cell lysis buffer (for 10 min) and AccuCheck Counting Beads (Invitrogen) to quantify circulating human T cell number as described [18, 19]. On day 30, human T cells in the peripheral blood of NSG mice were also analyzed by FACS with anti-human CD3 antibody (BD) before the mice were sacrified. All mice used were female (6–8 weeks old) purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained in compliance with an IACUC protocol approved by Emory University.
Data were shown as mean ± SEM. P values were calculated using the one-way analysis of variance test. P value of less than 0.05 was considered significant (* P < 0.05; ** P < 0.01; *** P < 0.001).
Human GIFT4 converts primary CLL B cells into antigen-presenting cell phenotype
STAT5/JAK signaling is essential for the conversion of CLL cells by GIFT4 treatment
GIFT4-primed CLL cells expand autologous T cells in vitro and in vivo
Human T cells primed by GIFT4-CLL cells are cytotoxic and kill primary CLL cells
In this study, we demonstrated that human GIFT4 can convert CLL B cells into immune-stimulatory helper cells, which function as APC and prime CLL-killing T cell response.
Primary CLL cells are monoclonal mature leukemic B cells that express CD5, CD19, CD20, IL-4 receptor (CD124), HLA-ABC and HLA-DR, but lack key T cell co-stimulatory molecules and adhesion molecules such as CD80, CD86, and CD54 . It was reported that CD40 ligation of CLL B cells with CD40 ligand (CD154)-transduced human embryonic fibroblast cells up-regulated CD54, CD80 and CD86, but not CD40 . In another study, transduction of adenovirus encoding chimeric CD154 augmented CLL cells to behave as APC . Untreated CLL cells express similar high levels of TLR9 as normal B cells . Activation of CLL cells with TLR9 ligand type B CpG oligodeoxynucleotides (CpG-ODN) resulted in significant increase of CD40, CD54, CD86, HLA-ABC and HLA-DR expression, but not CD80 . Treatment of CLL cells with combined CpG-ODN and IL-21 also enhanced the expression of CD54 and CD80, with slight increase of CD40 and CD86 on the cell surface, enabling CLL B cells functioned as APC-like cells .
Unlike primary CLL cells, CD40- or TLR9-ligated CLL cells, or CpG/IL-21 treated CLL cells, GIFT4-CLL cells robustly up-regulate the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54, which are likely essential surface factors for GIFT4-CLL cells functioning as APC to interact with T cells and prime T cell responses. Moreover GIFT4-CLL cells produce substantial amounts of IL-2, IL-8, FGFB, ICAM1, and IL-6, without significant production of GM-CSF, IFN-γ and CCL3. GIFT4-CLL cells are distinguished from our prior GIFT4-B cells that secrete GM-CSF and CCL3  and different from CD40/OX40-ligated CLL cells that produce IFN-γ , or CpG/IL-21 treated CLL cells that do not produce IL-2, ICAM-1, IL-6 and FGFB but secrete granzyme B . It has been reported that primary CLL cells produce CCL3 chemokine , however, we could not detect the chemokine in both untreated or GIFT4-treated CLL cells. Interesting, B cell receptor engagement with anti-IgM significantly enhanced chemokine CCL3 as well as CCL4 production by CLL cells . Collectively, our data showed that GIFT4-converted CLL cells possess a unique phenotype and secretome, which facilitates GIFT4-CLL cells to function as potent APC.
JAK/STAT signaling plays an important role in the survival and surface molecule expression of CLL B cells [14, 15, 33, 34]. CLL cells express both IL-4R and GM-CSFR. The binding of IL-4R by IL-4 activates JAK signaling , and leads to the phosphorylation of STAT1, STAT5, and STAT6 that enhances the survival of CLL cells [14, 34]. Unlike normal human B cells, CLL cells only express the GM-CSFR α, but not β subunit [15, 34, 35]. GM-CSFR α was showed to link with the activation of STAT3 and to promote the survival of CLL cells . GIFT4 has been previously shown to induce hyper phosphorylation of pan-STAT including STAT1, STAT3, STAT5 and STAT6 in normal B cells by clustering GM-CSFR and IL-4R on the cell surface and engagement of JAK1, JAK2 and JAK3 signaling . Indeed, we observed that GIFT4 stimulation also induced hyper phosphorylation of STAT5 in CLL cells, which is involved in upstream collaborative signaling complex of JAK1, JAK2 and JAK3. GIFT4-triggered JAK/STAT5 signaling further contributes to both the expression of co-stimulatory molecules CD80 and CD86, and the production of T cell-promoting cytokines IL-2 and IL-6 by GIFT4-CLL cells. However, we did not detect hyper phosphorylation of STAT1, STAT3 and STAT6 in CLL cells by GIFT4 exposure. Whether the lack of GM-CSFR β is linked to the absence of hyper phosphorylation of STAT1, STAT3 and STAT6 in CLL cells by GIFT4 stimulation remains to be determined.
Modified B cells have been utilized for cancer immunotherapy [36, 37]. Activated B cells acting as APC can elicit anti-tumor T cell response [11, 38, 39] or possess tumor-killing ability independently or through anti-tumor antibody production [40, 41]. Previous studies have also shown that in vitro modified CLL cells hyper-expressing adhesion molecules B7-1, ICAM-1 and LFA-3 , CD40 ligand  or both CD40 ligand and IL-2  may enhance effective T cell responses against CLL cells. Vaccination of whole CLL cells admixed with irradiated GM-CSF-secreting K562 bystander cells also promoted the expansion of IFN-γ+ leukemic-reactive T cells against CLL in patients after hematopoietic stem cell transplantation . GIFT4-CLL cells appear to have profound antigen presentation properties that may improve upon these approaches. GIFT4-converted CLL cells not only express immune co-stimulatory molecules CD40, CD80, CD86 and adhesion molecule ICAM-1 but also secrete immune-stimulatory cytokines IL-2 and IL-6, and have the potent ability to promote the expansion of autologous T cells. Those T cells produce cytotoxic factors such as IFN-γ, perforin, granzyme B, TRAIL, FAS ligand, CD314, and can directly kill primary CLL cells through perforin-mediated pathway. To our knowledge, this is the first report that primary CLL cells from subjects with CLL can be reprogrammed to anti-CLL immune helper cells.
GIFT4 fusokine has potent capability to reprogram CLL cells into APC-like effectors, expressing co-stimulatory molecules CD40, CD80, CD86, and adhesion molecules CD54. GIFT4-CLL cells secreted immune stimulatory cytokines IL-1β, IL-6, ICAM-1 and substantial IL-2, and prime autologous T cells into IFN-γ-producing CD314+ CLL-killer cells. The exclusive characteristics and the unique functions of GIFT4 and GIFT4-CLL cells support the notion that GIFT4 fusokine and GIFT4-CLL cells could be utilized as novel therapeutics for CLL immunotherapy.
JD designed and performed the experiment, analyzed and interpreted data, and wrote the manuscript; AP performed experiments and analyzed data; JCB collected human samples and critically reviewed the manuscript; YW performed experiments and analyzed data; SN performed experiments; JHW constructed the three-dimensional structure of GIFT4 protein. CRF collected human samples and critically reviewed the manuscript; JG designed the experiments; analyzed and interpreted data, wrote manuscript. All authors read and approved the final manuscript.
The authors thank Shala Yuan for laboratory technical support. This work was supported by NIH (5R01AI093881) and Georgia Cancer Coalition (JG); the Winship Robbins Scholar Award and the Developmental Fund of the Winship Cancer Center Support Grant (5P30CA138292-06) (JD).
The authors declare that they have no competing interests.
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