Pretreatment with IL-15 and IL-18 rescues natural killer cells from granzyme B-mediated apoptosis after cryopreservation.

Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

PVRL2 Suppresses Antitumor Immunity through PVRIG- and TIGIT-independent Pathways.

Poliovirus receptor-related 2 (PVRL2, also known as nectin-2 or CD112) is believed to act as an immune checkpoint protein in cancer; however, most insight into its role is inferred from studies on its known receptor, poliovirus receptor (PVR)-related immunoglobulin domain protein (PVRIG, also known as CD112R). Here, we study PVRL2 itself. PVRL2 levels were found to be high in tumor cells and tumor-derived exosomes. Deletion of PVRL2 in multiple syngeneic mouse models of cancer showed a dramatic reduction in tumor growth that was immune dependent. This effect was even greater than that seen with deletion of PD-L1. PVRL2 was shown to function by suppressing CD8+ T and natural killer cells in the tumor microenvironment. The loss of PVRL2 suppressed tumor growth even in the absence of PVRIG. In contrast, PVRIG loss showed no additive effect in the absence of PVRL2. T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade combined with PVRL2 deletion resulted in a near complete block in tumor growth. This effect was not recapitulated by the combined deletion of PVRL2 with its paralog, PVR, which is the ligand for TIGIT. These data uncover PVRL2 as a distinct inhibitor of the antitumor immune response with functions beyond that of its known receptor PVRIG. Moreover, the data provide a strong rationale for combinatorial targeting of PVRL2 and TIGIT for cancer immunotherapy.

CCR5 drives NK cell-associated airway damage in pulmonary ischemia-reperfusion injury.

Primary graft dysfunction (PGD) limits clinical benefit after lung transplantation, a life-prolonging therapy for patients with end-stage disease. PGD is the clinical syndrome resulting from pulmonary ischemia-reperfusion injury (IRI), driven by innate immune inflammation. We recently demonstrated a key role for NK cells in the airways of mouse models and human tissue samples of IRI. Here, we used 2 mouse models paired with human lung transplant samples to investigate the mechanisms whereby NK cells migrate to the airways to mediate lung injury. We demonstrate that chemokine receptor ligand transcripts and proteins are increased in mouse and human disease. CCR5 ligand transcripts were correlated with NK cell gene signatures independently of NK cell CCR5 ligand secretion. NK cells expressing CCR5 were increased in the lung and airways during IRI and had increased markers of tissue residency and maturation. Allosteric CCR5 drug blockade reduced the migration of NK cells to the site of injury. CCR5 blockade also blunted quantitative measures of experimental IRI. Additionally, in human lung transplant bronchoalveolar lavage samples, we found that CCR5 ligand was associated with increased patient morbidity and that the CCR5 receptor was increased in expression on human NK cells following PGD. These data support a potential mechanism for NK cell migration during lung injury and identify a plausible preventative treatment for PGD.

The CD16 and CD32b Fc-gamma receptors regulate antibody-mediated responses in mouse natural killer cells.

Natural killer (NK) cells are innate lymphocytes capable of mediating immune responses without prior sensitization. NK cells express Fc-gamma receptors (FcγRs) that engage the Fc region of IgG. Studies investigating the role of FcγRs on mouse NK cells have been limited due to lack specific reagents. In this study, we characterize the expression and biological consequences of activating mouse NK cells through their FcγRs. We demonstrate that most NK cells express the activating CD16 receptor, and a subset of NK cells also expresses the inhibitory CD32b receptor. Critically, these FcγRs are functional on mouse NK cells and can modulate antibody-mediated responses. We also characterized mice with conditional knockout alleles of Fcgr3 (CD16) or Fcgr2b (CD32b) in the NK and innate lymphoid cell (ILC) lineage. NK cells in these mice did not reveal any developmental defects and were responsive to cross-linking activating NK receptors, cytokine stimulation, and killing of YAC-1 targets. Importantly, CD16-deficient NK cells failed to induce antibody-directed cellular cytotoxicity of antibody-coated B-cell lymphomas in in vitro assays. In addition, we demonstrate the important role of CD16 on NK cells using an in vivo model of cancer immunotherapy using anti-CD20 antibody treatment of B-cell lymphomas.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

KLF12 Regulates Mouse NK Cell Proliferation.

NK cells are innate lymphocytes that play an integral role in tumor rejection and viral clearance. Unlike their other lymphocyte counterparts, NK cells have the unique ability to recognize and lyse target cells without prior exposure. However, there are no known NK cell-specific genes that are exclusively expressed by all NK cells. Therefore, identification of NK cell-specific genes would allow a better understanding of why NK cells are unique cytotoxic lymphocytes. From the Immunological Genome (ImmGen) Consortium studies, we identified kruppel-like factor 12 (Klf12), encoding a novel transcription factor, preferentially expressed in C57BL/6 mouse NK cells. KLF12 was dispensable for NK cell development, IFN-γ production, degranulation, and proliferation in Klf12 knockout mice. RNA-sequencing analysis revealed increased expression of Btg3, an antiproliferative gene, in KLF12-deficient NK cells compared with wild-type NK cells. Interestingly, competitive mixed bone marrow chimeric mice exhibited reduced development of KLF12-deficient NK cells, altered IFN-γ production and degranulation, and impairment of NK cell proliferation in vitro and in vivo in response to mouse CMV infection. KLF12-deficient NK cells from bone marrow chimeric mice also expressed higher levels of the IL-21R, which resulted in increased IL-21R signaling and correlated with greater inhibition of NK cell proliferation. Furthermore, IL-21 induced Btg3 expression, which correlated with arrested NK cell maturation and proliferation. In summary, we found that KLF12 regulates mouse NK cell proliferation potentially by regulating expression of Btg3 via IL-21.

Mouse cytomegalovirus-experienced ILC1s acquire a memory response dependent on the viral glycoprotein m12.

Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.

NKR-P1B expression in gut-associated innate lymphoid cells is required for the control of gastrointestinal tract infections.

Helper-type innate lymphoid cells (ILC) play an important role in intestinal homeostasis. Members of the NKR-P1 gene family are expressed in various innate immune cells, including natural killer (NK) cells, and their cognate Clr ligand family members are expressed in various specialized tissues, including the intestinal epithelium, where they may play an important role in mucosal-associated innate immune responses. In this study, we show that the inhibitory NKR-P1B receptor, but not the Ly49 receptor, is expressed in gut-resident NK cells, ILC, and a subset of γδT cells in a tissue-specific manner. ILC3 cells constitute the predominant cell subset expressing NKR-P1B in the gut lamina propria. The known NKR-P1B ligand Clr-b is broadly expressed in gut-associated cells of hematopoietic origin. The genetic deletion of NKR-P1B results in a higher frequency and number of ILC3 and γδT cells in the gut lamina propria. However, the function of gut-resident ILC3, NK, and γδT cells in NKR-P1B-deficient mice is impaired during gastrointestinal tract infection by Citrobacter rodentium or Salmonella typhimurium, resulting in increased systemic bacterial dissemination in NKR-P1B-deficient mice. Our findings highlight the role of the NKR-P1B:Clr-b recognition system in the modulation of intestinal innate immune cell functions.

The Inhibitory NKR-P1B:Clr-b Recognition Axis Facilitates Detection of Oncogenic Transformation and Cancer Immunosurveillance.

Natural killer (NK) cells express receptors specific for MHC class I (MHC-I) molecules involved in "missing-self" recognition of cancer and virus-infected cells. Here we elucidate the role of MHC-I-independent NKR-P1B:Clr-b interactions in the detection of oncogenic transformation by NK cells. Ras oncogene overexpression was found to promote a real-time loss of Clr-b on mouse fibroblasts and leukemia cells, mediated in part via the Raf/MEK/ERK and PI3K pathways. Ras-driven Clr-b downregulation occurred at the level of the Clrb (Clec2d) promoter, nascent Clr-b transcripts, and cell surface Clr-b protein, in turn promoting missing-self recognition via the NKR-P1B inhibitory receptor. Both Ras- and c-Myc-mediated Clr-b loss selectively augmented cytotoxicity of oncogene-transformed leukemia cells by NKR-P1B+ NK cells in vitro and enhanced rejection by WT mice in vivo Interestingly, genetic ablation of either one (Clr-b+/-) or two Clr-b alleles (Clr-b-/-) enhanced survival of Eµ-cMyc transgenic mice in a primary lymphoma model despite preferential rejection of Clr-b-/- hematopoietic cells previously observed following adoptive transfer into naïve wild-type mice in vivo Collectively, these findings suggest that the inhibitory NKR-P1B:Clr-b axis plays a beneficial role in innate detection of oncogenic transformation via NK-cell-mediated cancer immune surveillance, in addition to a pathologic role in the immune escape of primary lymphoma cells in Eµ-cMyc mice in vivo These results provide a model for the human NKR-P1A:LLT1 system in cancer immunosurveillance in patients with lymphoma and suggest it may represent a target for immune checkpoint therapy.Significance: A mouse model shows that an MHC-independent NK-cell recognition axis enables the detection of leukemia cells, with implications for a novel immune checkpoint therapy target in human lymphoma. Cancer Res; 78(13); 3589-603. ©2018 AACR.

Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition.

Natural killer (NK) cells are innate lymphocytes that aid in self-nonself discrimination by recognizing cells undergoing pathological alterations. The NKR-P1B inhibitory receptor recognizes Clr-b, a self-encoded marker of cell health downregulated during viral infection. Here, we show that Clr-b loss during mouse cytomegalovirus (MCMV) infection is predicated by a loss of Clr-b (Clec2d) promoter activity and nascent transcripts, driven in part by MCMV ie3 (M122) activity. In contrast, uninfected bystander cells near MCMV-infected fibroblasts reciprocally upregulate Clr-b expression due to paracrine type-I interferon (IFN) signaling. Exposure of fibroblasts to type-I IFN augments Clec2d promoter activity and nascent Clr-b transcripts, dependent upon a cluster of IRF3/7/9 motifs located ∼200 bp upstream of the transcriptional start site. Cells deficient in type-I IFN signaling components revealed IRF9 and STAT1 as key transcription factors involved in Clr-b upregulation. In chromatin immunoprecipitation experiments, the Clec2d IRF cluster recruited STAT2 upon IFN-α exposure, confirming the involvement of ISGF3 (IRF9/STAT1/STAT2) in positively regulating the Clec2d promoter. These findings demonstrate that Clr-b is an IFN-stimulated gene on healthy bystander cells, in addition to a missing-self marker on MCMV-infected cells, and thereby enhances the dynamic range of innate self-nonself discrimination by NK cells.

Genetic investigation of MHC-independent missing-self recognition by mouse NK cells using an in vivo bone marrow transplantation model.

MHC-I-specific receptors play a vital role in NK cell-mediated "missing-self" recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b(-/-) bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2D(b-/-) MHC-I-deficient BM cells. Selective rejection of Clr-b(-/-) BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b(-/-) BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1B(hi) NK cells, leaving the remaining NKR-P1B(lo) NK subset and MHC-I-dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b(-/-) hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I-deficient NK cells, Clr-b(-/-) NK cells were hyporesponsive to both NK1.1 (NKR-P1C)-stimulated and IL-12/18 cytokine-primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

Poxvirus infection-associated downregulation of C-type lectin-related-b prevents NK cell inhibition by NK receptor protein-1B.

Innate immune recognition of virus-infected cells includes NK cell detection of changes to endogenous cell-surface proteins through inhibitory receptors. One such receptor system is the NK cell receptor protein-1B (NKR-P1B) and its ligand C-type lectin-related-b (Clr-b). NKR-P1B and Clr-b are encoded within the NK cell gene complex, a locus that has been linked to strain-dependent differences in susceptibility to infection by poxviruses. In this study, we report the impact of vaccinia virus (VV) and ectromelia virus infection on expression of Clr-b and Clr-b-mediated protection from NK cells. We observed a loss of Clr-b cell-surface protein upon VV and ectromelia virus infection of murine cell lines and bone marrow-derived macrophages. The reduction of Clr-b is more rapid than MHC class I, the prototypic ligand of NK cell inhibitory receptors. Reduction of Clr-b requires active viral infection but not expression of late viral genes, and loss of mRNA appears to lag behind loss of Clr-b surface protein. Clr-b-mediated protection from NK cells is lost following VV infection. Together, these results provide the second example of Clr-b modulation during viral infection and suggest reductions of Clr-b may be involved in sensitizing poxvirus-infected cells to NK cells.