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Abstract
BackgroundThe peculiar multiple myeloma microenvironment, characterized by up-regulated levels of several inflammatory chemokines, including the CXCR3 receptor ligands CXCL9 and CXCL10, limits NK cell positioning into the bone marrow by interfering with CXCR4 function. It is still unclear if the consequent reduced influx of transferred cells into the tumor represents a potential limiting factor for the success of NK cell-based adoptive therapy. We hypothesize that inhibition of CXCR3 function on NK cells will result in increased tumor clearance, due to higher NK cell bone marrow infiltration.MethodsSince different activation protocols differently affect expression and function of homing receptors, we analyzed the bone marrow homing properties and anti-tumor efficacy of NK cells stimulated in vitro with two independent protocols. NK cells were purified from wild-type or Cxcr3−/− mice and incubated with IL-15 alone or with a combination of IL-12, IL-15, IL-18 (IL-12/15/18). Alternatively, CXCR3 function was neutralized in vivo using a specific blocking antibody. NK cell functional behavior and tumor growth were analyzed in bone marrow samples by FACS analysis.ResultsBoth activation protocols promoted degranulation and IFN-γ production by donor NK cells infiltrating the bone marrow of tumor-bearing mice, although IL-15 promoted a faster but more transient acquisition of functional capacities. In addition, IL-15-activated cells accumulated more in the bone marrow in a short time but showed lower persistence in vivo. Targeting of CXCR3 increased the bone marrow homing capacity of IL-15 but not IL12/15/18 activated NK cells. This effect correlated with a superior and durable myeloma clearance capacity of transferred cells in vivo.ConclusionsOur results demonstrate that in vitro activation affects NK cell anti-myeloma activity in vivo by regulating their BM infiltration. Furthermore, we provided direct evidence that CXCR3 restrains NK cell anti-tumor capacity in vivo according to the activation protocol used, and that the effects of NK cell-based adoptive immunotherapy for multiple myeloma can be improved by increasing their bone marrow homing through CXCR3 inhibition.
Highlights
The peculiar multiple myeloma microenvironment, characterized by up-regulated levels of several inflammatory chemokines, including the CXCR3 receptor ligands CXCL9 and CXCL10, limits Natural killer (NK) cell positioning into the bone marrow by interfering with CXCR4 function
To compare the bone marrow (BM)-homing ability of NK cells stimulated in different conditions with respect to migration to peripheral organs, we normalized their number in BM with that of the spleen, demonstrating that IL-12/15/18-stimulated cells were more prone to home to the BM than the IL-15-activated counterpart (Fig. 2b right panel)
Our results demonstrate that: i) in the BM of tumor-bearing mice, IL-15-activated NK cells have a numerical and functional advantage in the short time-frame compared to the IL-12/15/18 counterpart, allowing a superior tumor clearance capacity; ii) in vivo restimulation of donor NK cells with a single dose of IL-15 promotes long-term anti-tumor effect of NK cells preactivated with IL-12/15/18; iii) inhibition of CXCR3/ligand axes potentiates the ability to infiltrate BM and enhances anti-MM activity of IL-15 activated NK cell
Summary
The peculiar multiple myeloma microenvironment, characterized by up-regulated levels of several inflammatory chemokines, including the CXCR3 receptor ligands CXCL9 and CXCL10, limits NK cell positioning into the bone marrow by interfering with CXCR4 function. The use of IL-12/15/18-preactivated NK cells in combination with radiation therapy or with cytokine administration in vivo was shown to improve current protocols for immunotherapy of cancers in preclinical models and in clinical trials by sustaining their effector function and in vivo expansion [12, 18]. It is still unclear whether distinct stimulation protocols can differently modulate NK cell anti-tumor function by influencing their tissue homing properties
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