Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the ex vivo manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by ex vivo expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4. Reduced CXCR4 expression was associated with dampened in vitro NK cell migration toward its cognate ligand stromal-derived factor-1α (SDF-1α). NK cells isolated from patients with WHIM syndrome carry gain-of-function (GOF) mutations in CXCR4 (CXCR4 R334X ). Compared to healthy donors, we observed that NK cells expanded from WHIM patients have similar surface levels of CXCR4 but have a much stronger propensity to home to BM compartments when adoptively infused into NOD- scid IL2Rgamma null (NSG) mice. Therefore, in order to augment the capacity of adoptively infused NK cells to home to the BM, we genetically engineered ex vivo expanded NK cells to express the naturally occurring GOF CXCR4 R334X receptor variant. Transfection of CXCR4 R334X -coding mRNA into ex vivo expanded NK cells using a clinically applicable method consistently led to an increase in cell surface CXCR4 without altering NK cell phenotype, cytotoxic function, or compromising NK cell viability. Compared to non-transfected and wild type CXCR4-coding mRNA transfected counterparts, CXCR4 R334X -engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro . Importantly, expression of CXCR4 R334X on expanded NK cells resulted in significantly greater BM homing following adoptive transfer into NSG mice compared to non-transfected NK cell controls. Collectively, these data suggest up-regulation of cell surface CXCR4 R334X on ex vivo expanded NK cells via mRNA transfection represents a novel approach to improve homing and target NK cell-based immunotherapies to BM where hematological malignancies reside.
Regulatory T cells (Treg) suppress anti-tumor immune responses and their infiltration in the tumor microenvironment is associated with inferior prognosis in cancer patients. Thus, in order to enhance anti-tumor immune responses, selective depletion of Treg is highly desired. We found that treatment with zoledronic acid (ZA) resulted in a selective decrease in the frequency of Treg that was associated with a significant increase in proliferation of T cells and natural killer (NK) cells in peripheral blood of patients with metastatic cancer. In vitro, genome-wide transcriptomic analysis revealed alterations in calcium signaling pathways in Treg following treatment with ZA. Furthermore, co-localization of the nuclear factor of activated T cells (NFAT) and forkhead box P3 (FOXP3) was significantly reduced in Treg upon ZA-treatment. Consequently, reduced expression levels of CD25, STAT5 and TGFβ were observed. Functionally, ZA-treated Treg had reduced capacity to suppress T and NK cell proliferation and anti-tumor responses compared with untreated Treg in vitro. Treatment with ZA to selectively inhibit essential signaling pathways in Treg resulting in reduced capacity to suppress effector T and NK cell responses represents a novel approach to inhibit Treg activity in patients with cancer.
Objective. KIR and NKG2A receptors educate human NK cells to stay responsive to cells with diminished HLA class I. Here, we addressed whether the HLA class I-binding receptor LIR-1 (LILRB1/ ILT2/CD85j), which is widely expressed on human NK cells, can mediate education and contribute to antitumor functions of NK cells. Methods. Healthy donor NK cells either unstimulated, overnight cytokine-activated or ex vivo-expanded were used to target human cell lines. Phenotype and function were analysed using flow cytometry and 51 Cr-release assays. Results. We found that the inhibitory receptor LIR-1 can mediate NK cell education under specific conditions. This novel finding was exclusive to expanded NK cells and further characterisation of the cells revealed high expression of granzyme B and DNAM-1, which both previously have been linked to NK cell education. Corroborating the rheostat education model, LIR-1 co-expression with an educating KIR further increased the responsiveness of expanded NK cells. Inversely, antibody masking of LIR-1 decreased the responsiveness. LIR-1 + expanded NK cells displayed high intrinsic ADCC that, in contrast to KIR and NKG2A, was not inhibited by HLA class I. Conclusion. These findings identify a unique NK cell subset attractive for adoptive cell therapy to treat cancer. Given that LIR-1 binds most HLA class I molecules, this subset may be explored in both autologous and allogeneic settings to innately reject HLA class Itumor cells as well as HLA class I + target cells when combined with antitumor antibodies. Further studies are warranted to address the potential of this subset in vivo.
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