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Targeting of CXCR3 Improves Anti-Myeloma Efficacy of Adoptively Transferred Activated Natural Killer Cells

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Targeting of CXCR3 Improves Anti-Myeloma Efficacy of Adoptively Transferred Activated Natural Killer Cells

Valentina Bonanni et al. J Immunother Cancer.

Abstract

Background: 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. 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.

Methods: Since 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.

Results: Both 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.

Conclusions: Our 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.

Keywords: CXCR3; Cell migration; Chemokines; Multiple myeloma; Tumor immunotherapy.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Anti-MM efficacy and in vivo functional status of activated NK cells. Activated (5 × 105) CFSE+ NK cells obtained from splenocytes of C57BL/KaLwRij or PBS (No Cell) were i.v. transferred into MM-bearing mice 3 weeks after 5TGM1 cell injection. a) Tumor growth was determined by FACS analysis of CD138+ (tumor) cells among BM (2 tibias and femurs) and spleen cells at 48 h after transfer. The average ± SEM of 3 independent experiments with a total of at least 8 animals per group is shown. b) Activated NK cell functions in BM were determined by FACS analysis of CD107a + and IFN-γ + donor cell frequency 18 h and 48 h after transfer in MM-bearing mice. Graphs show average frequency ± SEM of CD107a + and IFN-γ + donor cells from 2 independent experiments, n = 5 per group. Time 0 corresponds to NK cell function immediately before the transfer. ND: Not detectable. Student t test was performed to compare no cell versus activated NK cell transferred mice (a) or differences between time 0 versus 18 h or 48 h (b). *p < 0.05; **p < 0.01
Fig. 2
Fig. 2
In vivo tissue migration and in vitro chemotaxis of activated NK cells. Activated CD45.1+ NK cells were mixed 1:1 with freshly isolated (naïve) CD45.2 NK cells, stained with CFSE and i.v. transferred into C57BL/KaLwRij mice 3 weeks after tumor cell injection. NK cell number was determined after 18 h in BM (two tibias and femurs), spleen and blood by FACS analysis of CD45.1+ or CD45.2+ NK1.1+ cells within donor CFSE+ cells and normalized with the number of input cells (% of input cells). a) Dot plots show the gating strategy for the analysis of IL-15 activated donor NK cells in the spleen of MM-bearing mice. b) CFSE+ cells were enumerated in each organ and frequency of donor cells out of transferred (input) cells is shown as average ± SEM of 2 independent experiments n = 5 mice per groups. Right hand graph: BM homing of activated donor NK cells was normalized by donor cell frequency into spleen. c) Tissue migration of activated NK cells in healthy control (ctrl) and tumor-bearing mice (tum). One-way ANOVA with multiple comparison was performed to compare tissue distribution of activated cells and naïve cells (b) and of activated cells in ctr and tum (c). *p < 0.05; **p < 0.01 d) In vitro chemotaxis assay of activated or control (cells treated with IL-15 10 ng/ml) NK cells in response to medium alone (no chemokine), to CXCL10 (250 ng/ml) or to CXCL12 (200 ng/ml). Results show average ± SEM from 3 independent experiments. One-way ANOVA was performed to compare migration of activated cells versus control cells. *p < 0.05; **p < 0.01
Fig. 3
Fig. 3
Expression of homing receptors on activated NK cells and NK cell migration in vitro. Purified NK cells were activated with IL-15, IL-12/15/18 for 20 h (control cells: IL-15 10 ng/ml). NK cell purity was assessed by anti-NK1.1 and -CD3 staining and expression of CXCR3, CXCR4, CD44 and CD49d (VLA-4) integrin chain was determined using specific antibodies. a) Upper panels show histogram plot of overlays of receptor staining in untreated and cytokine treated cells of a representative analysis. White filled histograms represent isotype control (i.c.) staining. Lower panels show average ± SEM median fluorescence intensity (MFI) from at least 3 independent analysis. Non-specific staining was subtracted from analysis. b) Detection of intracellular mRNA encoding for CXCR4 was done by PrimeFlow RNA Assay. c) Comparison between naïve and control cell receptor expression and migration: Left graphs show average ± SEM median fluorescence intensity (MFI) of CXCR3 and CXCR4 receptor. Right, in vitro chemotaxis assay in response to medium alone (no chemokine), to CXCL10 (250 ng/ml) or CXCL12 (200 ng/ml). Results show average ± SEM from 2 independent experiments
Fig. 4
Fig. 4
In vivo tissue distribution and anti-MM efficacy of IL-15 activated WT versus Cxcr3 deficient NK cells. a) Activated CFSE+ NK cells (4 × 105) composed of Cxcr3+/+ (CD45.1+) and Cxcr3−/− (CD45.2+) cells mixed 1:1 were transferred to tumor-bearing mice and donor cell number into tissues was quantified and normalized on input cells after 18 h. The number of transferred (donor) cells is shown in panel A as average ± SEM of frequency of input cell number. Two independent experiments with a total of at least 5 recipient mice per group were performed. b) Activated NK cells (5 × 105) from Cxcr3+/+ or Cxcr3−/− mice were transferred to MM-bearing mice and tumor burden was calculated after 48 h. Upper panel shows a representative analysis of frequency of CD138+ cells in the different conditions tested. Lower panel shows average ± SEM of frequency of tumor cells in BM from two independent experiments using a total of at least 6 animals per group. One-way ANOVA test was used to compare multiple groups. *p < 0.05;**p < 0.01; ***p < 0.005
Fig. 5
Fig. 5
Long term anti-MM efficacy of activated NK cells. Activated NK cells (5-6 × 105), obtained from splenocytes of C57BL/KaLwRij, or PBS (No Cell) were i.v. transferred into MM-bearing mice 3 weeks after 5TGM1 cell injection and IL-15 was administered 18 h later. a) Average ± SEM of frequency of tumor cells in BM and spleen 7 days after adoptive transfer (n = 5 in two independent experiments). One-way ANOVA test was used to compare multiple groups. b) BM persistence of transferred activated NK cells: IL-15 or IL-12/15/18 activated CFSE+ NK cells (4 × 105) were transferred to tumor-bearing mice and donor cells were enumerated in BM and spleen 1 and 7 days later. Upper left graph shows the number of donor cells as average ± SEM of frequency of input (transferred) cell number. Student t test was performed to analyze the difference between day 1 and day 7. Upper right graph shows average MFI ± SD of CXCR4 expression levels on donor NK cells 2 and 7 days after transfer (n = 3 per group; one experiment). Lower panels, 7 days after transfer, in vivo proliferation of transferred NK cells was analyzed by CFSE dilution. Histograms were gated on NK1.1+ transferred NK cells and one representative histogram from each group is shown (n = 3 per group). Numbers in the histogram plot indicate the percentage of cells that had proliferated. c) Average MFI ± SEM of CXCR3 and CXCR4 expression levels on activated NK cells cultured in vitro for 7 days in low IL-15 concentration (10 ng/ml). d) Endogenous NK cell number was determined in BM (two tibias and femurs) by FACS analysis of CD3-NK1.1+ cells in the CFSE population.*p < 0.05;**p < 0.01
Fig. 6
Fig. 6
Long term anti-MM efficacy of IL-15 activated NK cells after CXCR3 blockade. Activated NK cells (6 × 105) were i.v. transferred into MM-bearing mice 3 weeks after 5TGM1 cell injection with control hamster IgG or anti-CXCR3 blocking mAb (2 doses of 250 μg). Tumor growth was determined by FACS analysis of CD138+ (tumor) cells among BM cells 7 days after transfer. Lower graph: Activated NK cells were labeled with 2.5 μM CFSE and adoptively transferred in tumor-bearing mice and donor BM NK cell number was determined 7 days later by FACS analysis of CD3-NK1.1+ cells within donor CFSE+ cells and normalized with the number of input cells (% of input cells). Average number ± SEM of 2 independent experiments (n = 4 mice/group) is shown. Student t test was performed to compare control Ig versus anti-CXCR3 treated mice. **p < 0.01
Fig. 7
Fig. 7
Proposed model of NK cell function in BM upon adoptive cell therapy. Upper panels: NK cells activated with IL-15 infiltrate the BM and kill tumor cells with a faster kinetics than IL12/15/18 activated cells but their effect is more transient and is thus limited to a short time frame: upon initial activation of their anti-tumor function, IL-15 activated NK cells decrease their function and number starting from 48 h after transfer and their anti-tumor effect is no more evident after 7 days; on the other hand, IL-12/15/18 activated cells are poorly effective in the short time frame, this correlating with lower infiltration and slower activation of effector function than IL-15 activated cells. Nevertheless, they persist longer than IL-15 activated cells in BM and their anti-tumor effect becomes evident at 7 days after transfer. Lower panels: CXCR3 inhibition or genetic deletion increase activated IL-15 NK cell BM infiltration, thus improving and prolonging their anti-myeloma effect up to 7 days. Higher NK cell homing corresponds to improved engraftment in BM since transferred IL-15 NK cells persist up to 7 days. Conversely, IL-12/15/18 activated cells display long-term capacity to restrain tumor growth in vivo corresponding to better persistence in BM compared to IL-15 activated cells, but their BM infiltration and anti-tumor effect is not influenced by CXCR3

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