Redirecting NK cells to the lymph nodes to augment their lymphoma-targeting capacity.

CAR-NK cells can induce remission in lymphoma patients. We speculate that the full potential of adoptive NK cell immunotherapy against lymphoma is restricted by their poor lymph node (LN) homing capacity. Here, we have utilized a clinically approved transfection method with the aim of redirecting NK cells to LNs. Electroporation of ex vivo expanded NK cells with mRNAs coding for CCR7, CXCR5, and CD62L resulted in increased in vitro migration towards chemokines and mouse LN-derived supernatant. Following infusion into SCID/Beige mice, modified NK cells showed enhanced LN homing. Importantly, lymphoma patient-derived NK cells were equally well expanded and engineered as healthy donor NK cells, highlighting their translational potential. Additionally, the introduction of high-affinity CD16, together with the homing molecules, also augmented their ADCC capacity against autologous lymphoma cells. Hence, genetic engineering can be utilized to enhance NK cell LN homing. The homing concept may synergize with CAR- or monoclonal/bi-/tri-specific antibody-based approaches.

KIR2DS1 and KIR2DL1-C245 Dominantly Repress NK Cell Degranulation Triggered by Monoclonal or Bispecific Antibodies, whereas Education by Uptuning Inhibitory Killer Ig-related Receptors Exerts No Advantage in Ab-dependent Cellular Cytotoxicity.

NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.

LIR-1 educates expanded human NK cells and defines a unique antitumor NK cell subset with potent antibody-dependent cellular cytotoxicity.

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 51Cr-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 I- tumor 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.

CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy.

Natural killer (NK) cells are cytotoxic lymphocytes of our immune system with the ability to identify and kill certain virally infected and tumor-transformed cells. During the past 15 years, it has become increasingly clear that NK cells are involved in tumor immune surveillance and that they can be utilized to treat cancer patients. However, their ability to induce durable responses in settings of adoptive cell therapy needs to be further improved. One possible approach is to genetically engineer NK cells to augment their cytotoxicity per se, but also their ability to persist in vivo and home to the tumor-bearing tissue. In recent years, investigators have explored the potential of viral transduction and mRNA electroporation to modify NK cells. Although these methods have generated promising data, they are associated with certain limitations. With the increasing advances in the CRISPR/Cas9 technology, investigators have now turned their attention toward using this technology with NK cells as an alternative method. In this book chapter, we introduce NK cells and provide an historical overview of techniques to genetically engineer lymphocytes. Further, we elucidate protocols for inducing double-strand breaks in NK cells via CRISPR/Cas9 together with readouts to address its efficacy and functional outcome. We also discuss the pros and cons of the described readouts. The overall aim of this book chapter is to help introduce the CRISPR/Cas9 technology to the broader audience of NK cell researchers.

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4R334X.

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 (CXCR4R334X). 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 IL2Rgammanull (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 CXCR4R334X receptor variant. Transfection of CXCR4R334X-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, CXCR4R334X-engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro. Importantly, expression of CXCR4R334X 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 CXCR4R334X 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.

Zoledronic acid inhibits NFAT and IL-2 signaling pathways in regulatory T cells and diminishes their suppressive function in patients with metastatic cancer.

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 TGFß 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.