Engineered Fusokine GIFT4 Licenses the Ability of B Cells to Trigger a Tumoricidal T-cell Response

Abstract

Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL-4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL-4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Further, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B cell-deficient mice, confirming a B cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy.

GM-CSF and IL-4 Fusion Cytokine Induces B Cell-Dependent Hematopoietic Regeneration

“…45 Murine GIFT4-B cells produce an array of hematopoietic cytokines and growth factors, including IL-1, IL-6, GM-CSF, VEGF, and chemokine CCL3, but not G-CSF and SCF. 28 The role of CCL3 on hematopoiesis is controversial. In the presence of GM-CSF or M-CSF or in combination with SCF, IL-3, IL-6, and Flt3L, CCL3 has a stimulatory effect on the expansion and proliferation of hematopoietic progenitor cells.…”

“…28 It was reported that GM-CSF promoted hematopoietic recovery by enhancing the survival of HSCs and the proliferation and differentiation of myeloid lineage cells in irradiated mice. 48,49 Cytokines IL-1 and IL-12 also have pro-hematopoietic effects, enhancing hematopoiesis and immune reconstitution following irradiation.…”

“…27 Recently, we generated a synthetic fusokine that is derived from the fusion of GM-CSF and IL-4 (named as GIFT4) and found that GIFT4 protein has a potent immune stimulatory property and educates naive B cells into B effector cells with anti-tumor activity. 28 Unexpectedly, we discovered that GIFT4 triggers a novel B cell function on hematopoiesis in vivo. Here, we show that GIFT4 treatment expands endogenous LSK cells and its subpopulations, HSCs and multipotent progenitors (MPPs), in a B cell-dependent manner and robustly accelerates long-term hematopoietic reconstitution following myeloablative therapy in mice.…”

“…28 Surprisingly, delivery of GIFT4 protein into naive C57BL/6J (B6) mice significantly increased bone marrow cellularity ( Figure S1) and the proportion of endogenous LSK cells in the Lin-negative population of bone marrow cells in comparison with GM-CSF and IL-4 treatment or PBS controls, as determined by fluorescence-activated cell sorting (FACS) assay ( Figure 1B). The percentage of LSK cells in the femur of GIFT4-treated mice was 5.8% on average, 2.5-fold higher than the ones in GM-CSF and IL-4-or PBS-treated mice (2.3% and 2.2% on average, respectively) ( Figure 1C).…”

“…GIFT4-B cells, as well as purified naive B cells, are absent of LSK cells (Figure S6B) and express a panel of B effector markers ( Figure S6C), as we previously showed. 28 GFP-positive GIFT4-B cells have the capability to migrate into the bone marrow and spleen, but not the brain, in vivo ( Figures S6C and S6D). The measurement of body weight showed that the delivery of GIFT4-B cells prevented mice from irradiation-induced body weight loss ( Figure 5A); the mice continued to gain weight and survived similarly to bone marrow-transferred mice ( Figure 5B).…”