Natural Killer Response Research Articles

Polygenic polymorphism is associated with NKG2A repertoire and influences lymphocyte phenotype and function

AbstractCD94/NKG2A is a heterodimeric receptor commonly found on natural killer (NK) and T cells, and its interaction with its ligand HLA-E on adjacent cells leads to inhibitory signaling and cell suppression. We have identified several killer cell lectin–like receptor (KLR)C1 (NKG2A) single nucleotide polymorphisms (SNPs) that are associated with NKG2A expression on NK cells, CD8+ T cells, and Vγ9/Vδ2+ T cells. Additionally, due to strong linkage disequilibrium, polymorphisms in KLRC2 (NKG2C) and KLRK1 (NKG2D) are also associated with NKG2A surface density and frequency. NKG2A surface expression correlates with single-cell NK responsiveness, and NKG2A+ NK cell frequency is associated with total NK repertoire response and inhibitability, making the identification of SNPs responsible for expression and frequency important for predicting the innate immune response. Because HLA-E expression is dependent on HLA class I signal peptides, we analyzed the relationship between peptide abundance and HLA-E expression levels. Our findings revealed a strong association between peptide availability and HLA-E expression. We identified the HLA-C killer immunoglobulin–like receptor ligand epitope as a predictive marker for HLA-ABC expression, with the HLA-C1 epitope associated with high HLA-E expression and the HLA-C2 epitope associated with low HLA-E expression. The relationship between HLA-C epitopes and HLA-E expression was independent of HLA-E allotypes and HLA-B leader peptides. Although HLA-E expression showed no significant influence on NKG2A-mediated NK education, it did affect NK cell inhibition. In summary, these findings underscore the importance of NKG2A SNPs and HLA-C epitopes as predictive markers of NK cell phenotype and function and should be evaluated as prognostic markers for diseases that express high levels of HLA-E.

The KIR-HLA-CD16a Immunogenetic Profile Influences NK Cell-Mediated ADCC and Response to Rituximab Therapy in B-Cell Lymphomas

Rituximab-based chemoimmunotherapy (R-CHOP) is the standard of care in most B-cell lymphomas, however there is marked interpatient heterogeneity in treatment response. An important mechanism of Rituximab (anti-CD20) action is through natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but the factors influencing this are poorly understood. Here, we present findings from a preclinical study investigating the role of NK cell receptor polymorphisms and HLA allelic variation on ADCC against aggressive B-cell lymphoma cell lines. Genotyping was conducted using Luminex-based SSO and Sanger sequencing. Multicolor flow cytometry was used to assess the response of activated NK cells from healthy donors against 8 lymphoma cell lines. Individuals carrying the GG or GT allele in CD16a were found to have a 3-8 fold greater NK cell response compared to individuals with a TT allele and rituximab-mediated ADCC was significantly greater in individuals with a KIR-3DL1 allele when tested against B-cell lymphomas lacking the HLA-Bw4 allele. An additive effect was observed when considering both CD16a and KIR-HLA together. As expected, CD20 expression was a strong correlate of NK cell response. In summary, NK cell-mediated ADCC in response to ADCC appears to be influenced by the KIR-HLA-CD16a genotype. These findings suggest a potential role for immunogenotyping in B-cell lymphomas treated with Rituximab. Additionally, the presence of the shared F c receptor domain opens the possibility of applying this framework towards predicting NK cell responses to antibody therapies in other diseases.

Abstract 4263: Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer

Abstract High grade serous ovarian cancer (OC) and ovarian carcinosarcoma, referred as “Cold” tumors have restricted treatment choices and being associated with high mortality. Current immune checkpoint inhibitors (CPIs) have had modest impact for OC treatment; with anti PD-1/PD-L1 single therapies reported response rates of 4-15%. Accordingly, additional approaches are important for these patients. Natural killer (NK) cells represent an important subset of effector lymphocytes with strategic approaches for maximizing NK reactivity to target OC are likely important. Of interest, an inhibitory receptor and glyco immune checkpoint- Siglec 7 is expressed on 100% of peripheral blood and umbilical cord NK cells. We have generated antibodies against Siglec 7 through an optimized human Siglec 7 DNA/protein immunization approach in humanized next generation transgenic mice. By high throughput flow screening, we identified several Siglec 7 antibodies and down selected relevant clones. A unique clone DB7.2 was highly specific for Siglec 7. We demonstrated the ability of DB7.2 (human IgG1) to activate NK cells and induce OC cell killing using xCELLigence RTCA for in vivo OC challenge studies. DB7.2 (1μg/ml) bound to >90% of NK cells including both CD56dim and CD56bright subsets, evaluated in the PBMCs of multiple donors. It showed strong binding to recombinant Siglec 7 and Siglec 7 transduced HEK293T but not to wild type 293T cells supporting specificity. DB7.2 induced specific killing of multiple OC lines (OVCAR3&10,TOV21G, CaOV3, OVISE, PEO4) carrying different mutations; BRCA1&2, AKT, TP53, PIK3CA, BRAF etc. and resistant against a wide array of cancer drug targets; HSP90, HDAC, MTORC, DNA alkylating agents, EGFR, PARP, PI3K, and WEE1. Tumor killing was indicated to be mediated via enhanced secretion of soluble Fas, perforin, granulysin as well as granzyme A. Of note, OVISE (BRCA1mutated) and PEO4 (BRCA2 mutated, PARPi resistant) were susceptible to DB7.2 killing with EC50- 82.67 and 68.67 nM respectively. A single dose of in vivo expressed DB7.2 significantly reduced the tumor burden in an OVISE challenged humanized mice model enhancing median survival by 57 days. As OCs are highly diverse in nature and likely to require combinatorial approaches for simultaneous targeting of immune pathways; we combined DB7.2 with anti-PD1 for further investigation. In xCELLigence assay, anti-PD1 demonstrated killing of PEO4 cells with EC50 680 nM. The combination of anti PD-1 with DB7.2 showed further enhancement of OC killing in the presence of human PBMCs. This is the first demonstration of the impact of Siglec 7 targeting mAb alone as well as in combination with anti-PD1 (NK and T cell CPIs) studied for targeting OC or any human tumor. These studies have important implications for tumor therapy and provide a novel non T cell CPI potential approach to augment current immune therapy strategies. Citation Format: Devivasha Bordoloi, Abhijeet J. Kulkarni, Opeyemi S. Adeniji, M Betina Pampena, Pratik S. Bhojnagarwala, Shushu Zhao, Elizabeth M. Parzych, Rugang Zhang, Michael R. Betts, Mohamed Abdel-Mohsen, David B. Weiner. Anti-Siglec 7 antibody displays potent anti-tumor immunity and demonstrates improved tumor control in combination with anti-PD1 in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4263.

Transcriptional reprogramming of natural killer cells by vaccinia virus shows both distinct and conserved features with mCMV.

Natural killer (NK) cells have an established role in controlling poxvirus infection and there is a growing interest to exploit their capabilities in the context of poxvirus-based oncolytic therapy and vaccination. How NK cells respond to poxvirus-infected cells to become activated is not well established. To address this knowledge gap, we studied the NK cell response to vaccinia virus (VACV) in vivo, using a systemic infection murine model. We found broad alterations in NK cells transcriptional activity in VACV-infected mice, consistent with both direct target cell recognition and cytokine exposure. There were also alterations in the expression levels of specific NK surface receptors (NKRs), including the Ly49 family and SLAM receptors, as well as upregulation of memory-associated NK markers. Despite the latter observation, adoptive transfer of VACV-expercienced NK populations did not confer protection from infection. Comparison with the NK cell response to murine cytomegalovirus (MCMV) infection highlighted common features, but also distinct NK transcriptional programmes initiated by VACV. Finally, there was a clear overlap between the NK transcriptional response in humans vaccinated with an attenuated VACV, modified vaccinia Ankara (MVA), demonstrating conservation between the NK response in these different host species. Overall, this study provides new data about NK cell activation, function, and homeostasis during VACV infection, and may have implication for the design of VACV-based therapeutics.

TLR8 agonist partially improves IFN-γ deficiency of NK cells in chronic hepatitis B through the synergy of monocytes.

Natural killer (NK) cells exhibit a selective deficiency of IFN-γ production in chronic hepatitis B (CHB). Toll-like receptor 8 (TLR8) agonists could induce IFN-γ production in immune cells, although their effects on the deficiency in NK cells remain unclear. To investigate TLR8 expression in NK cells and the effect of TLR8 agonists in patients with CHB METHODS: We enrolled 32 patients with CHB and 19 healthy controls to assess TLR8 expression and IFN-γ production in NK cells. The sorted NK cells and monocytes were co-cultured to compare the extent of IFN-γ and IL-10 production after TLR8 agonist ssRNA40 stimulation. The synergic effect of NK cells and monocytes was assessed by blocking IL-12 and IL-18. We recruited another22 patients with CHB undergoing nucleotide analogue (NA) therapy to explore the impact of antiviral treatment on the ssRNA40-mediated response of NK cells. In patients with CHB, TLR8 expression in NK cells was up-regulated, accompanied by insufficient IFN-γ production. The enhanced IFN-γ secretion by ssRNA40 in NK cells depended on monocyte-derived IL-12 and IL-18. NK cells displayed an imbalanced response to ssRNA40 in patients with CHB with a weak increase in IFN-γ despite a higher IL-10 production. The response was improved in patients with CHB undergoing NA therapy. In patients with CHB, targeting TLR8 partially rescues the IFN-γ insufficiency in NK cells.However, NK cells show an inhibitory response to TLR8 agonist stimulation. TLR8 agonist combined with NA may enhance the antiviral effect of NK cells.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P < 0.001) and secretion of IFN-gamma (P < 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P < 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P < 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (>85%) and viability (>80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P < 0.001) and 2 times more potent than s15 NK cells (P < 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P < 0.05) or s15 NK cells (P < 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P < 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P < 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

Role of Early Life Cytotoxic T Lymphocyte and Natural Killer Cell Immunity in Paediatric HIV Cure/Remission in the Anti-Retroviral Therapy Era.

Only three well-characterised cases of functional cure have been described in paediatric HIV infection over the past decade. This underlines the fact that early initiation of combination antiretroviral therapy (cART), whilst minimising the size of the viral reservoir, is insufficient to achieve cure, unless other factors contribute. In this review, we consider these additional factors that may facilitate functional cure in paediatric infection. Among the early life immune activity, these include HIV-specific cytotoxic T-lymphocyte (CTL) and natural killer (NK) cell responses. The former have less potent antiviral efficacy in paediatric compared with adult infection, and indeed, in early life, NK responses have greater impact in suppressing viral replication than CTL. This fact may contribute to a greater potential for functional cure to be achieved in paediatric versus adult infection, since post-treatment control in adults is associated less with highly potent CTL activity, and more with effective antiviral NK cell responses. Nonetheless, antiviral CTL responses can play an increasingly effective role through childhood, especially in individuals expressing then ‘protective’ HLA-I molecules HLA-B*27/57/58:01/8101. The role of the innate system on preventing infection, in shaping the particular viruses transmitted, and influencing outcome is discussed. The susceptibility of female fetuses to in utero mother-to-child transmission, especially in the setting of recent maternal infection, is a curiosity that also provides clues to mechanisms by which cure may be achieved, since initial findings are that viral rebound is less frequent among males who interrupt cART. The potential of broadly neutralising antibody therapy to facilitate cure in children who have received early cART is discussed. Finally, we draw attention to the impact of the changing face of the paediatric HIV epidemic on cure potential. The effect of cART is not limited to preventing AIDS and reducing the risk of transmission. cART also affects which mothers transmit. No longer are mothers who transmit those who carry genes associated with poor immune control of HIV. In the cART era, a high proportion (>70% in our South African study) of transmitting mothers are those who seroconvert in pregnancy or who for social reasons are diagnosed late in pregnancy. As a result, now, genes associated with poor immune control of HIV are not enriched in mothers who transmit HIV to their child. These changes will likely influence the effectiveness of HLA-associated immune responses and therefore cure potential among children.

Comprehensive Analysis of RAPGEF2 for Predicting Prognosis and Immunotherapy Response in Patients with Hepatocellular Carcinoma.

Background Hepatocellular carcinoma (HCC) is the sixth most common tumor worldwide. Additionally, deletion of RAPGEF2 plays a critical role in CNV and related to tumor immune microenvironment, whereas the prognostic potential of RAPGEF2 in HCC patient needs to be explored. Methods We looked for prognostic potential genes in HCC using a variety of R programs. Then, using the LASSO Cox regression, we thoroughly evaluated and integrated the RAPGEF2-related genes from TCGA database. Meanwhile, utilizing TCGA and ICGA databases, the link between RAPGEF2 and immunotherapy response in HCC was studied. In vivo, the effect of RAPGEF2 on tumor development and the capacity of natural killer (NK) cells to recruit were confirmed. To ascertain the connection between RAPGEF2-related genes and the prognosis of HCC, a prognostic model was created and validated. Result We demonstrated RAPGEF2 has a differential expression, and patients with deletion of RAPGEF2 gene get shorter survival in HCC. Additionally, the tissues without RAPGEF2 have a weaker ability to recruit the NK cells and response to immunotherapy. After that, we scoured the database for eight RAPGEF2-related genes linked with a better prognosis in HCC patients. Additionally, silencing RAPGEF2 accelerated tumor development in the HCC mouse model and decreased CD56+ NK cell recruitment in HCC tissues. TCGA database was used to classify patients into low- and high-risk categories based on the expression of related genes. Patients in the low-risk group had a significantly greater overall survival than those in the high-risk group (P < 0.001). Meanwhile, the low-risk group demonstrated connections with the NK cell and immunotherapy response. Finally, the prognostic nomogram showed a high sensitivity and specificity for predicting the survival of HCC patients at 1, 2, and 3 years. Conclusion The prognostic model based on RAPGEF2 and RAPGEF2-related genes showed an excellent predictive performance in terms of prognosis and immunotherapy response in HCC, therefore establishing a unique prognostic model for clinical assessment of HCC patients.

Oncolytic Orf virus licenses NK cells via cDC1 to activate innate and adaptive antitumor mechanisms and extends survival in a murine model of late-stage ovarian cancer

BackgroundNovel therapies are needed to improve outcomes for women diagnosed with ovarian cancer. Oncolytic viruses are multifunctional immunotherapeutic biologics that preferentially infect cancer cells and stimulate inflammation with the potential...

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection.

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4-14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.

Role of Toll‑like receptors in natural killer cell function in acute lymphoblastic leukemia (Review)

Natural killer (NK) cells are specialized lymphocytes primarily involved in the response to infection and tumors. NK cells are characterized by the presence of specific surface molecules, as well as a wide repertoire of receptors that impart microenvironment-dependent effector functions. Among these receptors, Toll-like receptors (TLRs) can be activated to condition the NK response to either a cytotoxic or immunoregulatory phenotype. However, cellular function is frequently impaired during disorders such as cancer. In the last decade, it has become increasingly evident that the stimulation of NK cells is a requirement for their increased cytotoxic activity. TLR activation has been suggested as an alternative route for reestablishing the antitumor activity of NK cells. The present review summarizes the characteristics of NK cells, their receptors, the expression and function of NK cell TLRs, and their functional status in cancer, primarily acute lymphoblastic leukemia.

Licensing Natural Killers for Antiviral Immunity.

Immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors (IRs) enable discrimination between self- and non-self molecules on the surface of host target cells. In this regard, they have a vital role in self-tolerance through binding and activating intracellular tyrosine phosphatases which can inhibit cellular activation. Yet, self-MHC class I (MHC I)-specific IRs are versatile in that they can also positively impact lymphocyte functionality, as exemplified by their role in natural killer (NK) cell education, often referred to as ’licensing‘. Recent discoveries using defined mouse models of cytomegalovirus (CMV) infection have revealed that select self-MHC I IRs can increase NK cell antiviral defenses as well, whereas other licensing IRs cannot, or instead impede virus-specific NK responses for reasons that remain poorly understood. This review highlights a role for self-MHC I ‘licensing’ IRs in antiviral immunity, especially in the context of CMV infection, their impact on virus-specific NK cells during acute infection, and their potential to affect viral pathogenesis and disease.

Modulating the Immunologic Microenvironment in AML By Blocking Both CXCR4 and PD-L1

BackgroundWe have shown the importance of microenvironments by displaying the restoration of dysfunctional natural killer (NK) cells' antitumor effects both in vitro and in vivo after a trial of CXCR4 inhibition combined with conventional chemotherapy in acute myeloid leukemia (AML). Recently, the inhibition of programmed death-1(PD-1) /PD-ligand1 (PD-L1) axis in solid cancer has been regarded as a key factor to treating cancer by activating the suppressed immune cells. However, the significance of PD-1/PD-L1 expression in AML has yet to be established, even though PD-1/PD-L1 has already been targeted in clinical trials. Thus, our aim was to revitalize suppressed immune cells by utilizing CXCR4 blockade and anti-PD-L1 in combination with chemotherapy in an AML mouse model.Methods and Results The leukemic mouse model was induced by injecting C1498 (murine AML cell line) cells into C57/Bl-6 mice at D0, and leukemic blasts of the organs of several subgroups treated with cytosine arabinoside (Ara-C) (2 mg/kg, I.P.), plerixafor (2.5 mg/kg, S.C.), and anti-PD-L1 (200 ug/kg, I.P.) in one-by-one additive manners were examined (Fig. 1). Flow cytometric analyses at D+3, D+14, D+19, D+26 and histologic examination after sacrifice were performed to read the therapy-responsive changes of immune cell subsets and blasts reduction. Our data indicated noticeable benefits of this triple combinational treatment with plerixafor, anti-PD-L1, and Ara-C (AMP subgroup in Fig. 2 & Fig. 3) in removal of blasts in vivo; especially, survival rates of leukemic mice in the triple-combined treatment subgroup were higher than those in other subgroups. Moreover, in immune cell subsets in the peripheral blood, the frequency of CD4+ and CD8+ T cells was significantly increased specifically early after completion of the treatment in the triple combinational subgroup with remarkable changes of monocytic myeloid-derived suppressor cells (M-MDSCs) as well as regulatory T cells (Tregs), compared to that of other subgroups. Interestingly, in the triple combinational subgroup, Tregs and M-MDSCs were both significantly decreased while G-MDSCs were significantly increased in the bone marrow (BM) of leukemic mice that were sacrificed alive at D+26. In addition, the phenotypic levels of PD-1 both in the BM and the spleen were relatively low compared to those of other subgroups. These findings implied that the modulation of immune status by applying plerixafor and anti-PD-L1 with Ara-C could lead to a more efficient leukemic blasts ablation through the activation of CD4+ and CD8+ T cells and possibly the suppression of Tregs and/or M-MDSCs in AML. Integrated responses of NK, NKT, and DC cells should be pursued in the future.Conclusion We demonstrated the impact of a strategic triple combinational therapy with anti-PD-L1 and plerixafor with Ara-C in AML. Our results suggest some clues and provide guidelines to developing therapeutic strategies for chemotherapy-resistant patients in clinic by the modulation of leukemic microenvironments. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Natural Killer Cells Recruitment in Oncolytic Virotherapy: A Mathematical Model.

In this paper, we investigate how natural killer (NK) cell recruitment to the tumor microenvironment (TME) affects oncolytic virotherapy. NK cells play a major role against viral infections. They are, however, known to induce early viral clearance of oncolytic viruses, which hinders the overall efficacy of oncolytic virotherapy. Here, we formulate and analyze a simple mathematical model of the dynamics of the tumor, OV and NK cells using currently available preclinical information. The aim of this study is to characterize conditions under which the synergistic balance between OV-induced NK responses and required viral cytopathicity may or may not result in a successful treatment. In this study, we found that NK cell recruitment to the TME must take place neither too early nor too late in the course of OV infection so that treatment will be successful. NK cell responses are most influential at either early (partly because of rapid response of NK cells to viral infections or antigens) or later (partly because of antitumoral ability of NK cells) stages of oncolytic virotherapy. The model also predicts that: (a) an NK cell response augments oncolytic virotherapy only if viral cytopathicity is weak; (b) the recruitment of NK cells modulates tumor growth; and (c) the depletion of activated NK cells within the TME enhances the probability of tumor escape in oncolytic virotherapy. Taken together, our model results demonstrate that OV infection is crucial, not just to cytoreduce tumor burden, but also to induce the stronger NK cell response necessary to achieve complete or at least partial tumor remission. Furthermore, our modeling framework supports combination therapies involving NK cells and OV which are currently used in oncolytic immunovirotherapy to treat several cancer types.

Transcriptional and epigenetic regulation of memory NK cell responses.

Natural killer (NK) cells are cytotoxic innate lymphocytes with key roles in host protection against viruses and malignancy. Notwithstanding their historical classification as innate immune cells, NK cells are now understood to have some capacity to mount memory or memory-like immune responses in which effector cells undergo antigen-driven expansion and give rise to long-lived memory cells with enhanced functionality.Understanding how antigen-specific effector and memory NK responses are regulated is an important and active area of research in the field. Here, we discuss key transcription factors and epigenetic processes involved in antigen-specific effector and memory NK cell differentiation.

From Chickens to Humans: The Importance of Peptide Repertoires for MHC Class I Alleles.

In humans, killer immunoglobulin-like receptors (KIRs), expressed on natural killer (NK) and thymus-derived (T) cells, and their ligands, primarily the classical class I molecules of the major histocompatibility complex (MHC) expressed on nearly all cells, are both polymorphic. The variation of this receptor-ligand interaction, based on which alleles have been inherited, is known to play crucial roles in resistance to infectious disease, autoimmunity, and reproduction in humans. However, not all the variation in response is inherited, since KIR binding can be affected by a portion of the peptide bound to the class I molecules, with the particular peptide presented affecting the NK response. The extent to which the large multigene family of chicken immunoglobulin-like receptors (ChIRs) is involved in functions similar to KIRs is suspected but not proven. However, much is understood about the two MHC-I molecules encoded in the chicken MHC. The BF2 molecule is expressed at a high level and is thought to be the predominant ligand of cytotoxic T lymphocytes (CTLs), while the BF1 molecule is expressed at a much lower level if at all and is thought to be primarily a ligand for NK cells. Recently, a hierarchy of BF2 alleles with a suite of correlated properties has been defined, from those expressed at a high level on the cell surface but with a narrow range of bound peptides to those expressed at a lower level on the cell surface but with a very wide repertoire of bound peptides. Interestingly, there is a similar hierarchy for human class I alleles, although the hierarchy is not as wide. It is a question whether KIRs and ChIRs recognize class I molecules with bound peptide in a similar way, and whether fastidious to promiscuous hierarchy of class I molecules affect both T and NK cell function. Such effects might be different from those predicted by the similarities of peptide-binding based on peptide motifs, as enshrined in the idea of supertypes. Since the size of peptide repertoire can be very different for alleles with similar peptide motifs from the same supertype, the relative importance of these two properties may be testable.

Epstein\u2013Barr virus peptides derived from latent cycle proteins alter NKG2A\u2009+\u2009NK cell effector function

Natural killer (NK) cells control viral infection through the interaction between inhibitory receptors and human leukocyte antigen (HLA) ligands and bound peptide. NK cells expressing the inhibitory receptor NKG2A/CD94 recognize and respond to autologous B cells latently infected with Epstein–Barr virus (EBV). The mechanism is not yet understood, thus we investigated peptides derived from seven latent proteins of EBV in the interaction of NKG2A and its ligand HLA-E. Functional analysis demonstrated that EBV peptides can bind to HLA-E and block inhibition of NK cell effector function. Moreover, analysis of DNA from 79 subjects showed sequence variations in the latent protein, LMP1, which alters NK responses to EBV. We provide evidence that peptides derived from EBV latent cycle proteins can impair the recognition of NKG2A despite being presented by HLA-E, resulting in NK cell activation.

BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis

BackgroundTargeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine–threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in...

Natural Killer Cells: Tumor Surveillance and Signaling.

Natural killer (NK) cells play a pivotal role in cancer immunotherapy due to their innate ability to detect and kill tumorigenic cells. The decision to kill is determined by the expression of a myriad of activating and inhibitory receptors on the NK cell surface. Cell-to-cell engagement results in either self-tolerance or a cytotoxic response, governed by a fine balance between the signaling cascades downstream of the activating and inhibitory receptors. To evade a cytotoxic immune response, tumor cells can modulate the surface expression of receptor ligands and additionally, alter the conditions in the tumor microenvironment (TME), tilting the scales toward a suppressed cytotoxic NK response. To fully harness the killing power of NK cells for clinical benefit, we need to understand what defines the threshold for activation and what is required to break tolerance. This review will focus on the intracellular signaling pathways activated or suppressed in NK cells and the roles signaling intermediates play during an NK cytotoxic response.

Abstract B43: Positron emission tomography (PET) imaging of the natural killer (NK) cell activation receptor NKp30

Abstract Natural killer (NK) cells are essential defenders against viral and cancer threats. Therapeutic strategies utilizing NK cells include both adoptive cell transfer and modulating NK response in situ. Therapeutic response is gauged using CT/MRI or analysis of circulating NK cells via flow cytometry. Measuring tumor size via CT/MRI has limited prognostic value for immunotherapies and circulating NK cell activity is a poor surrogate for NK cell engagement and activation at desired tumor sites. New approaches are needed to noninvasively monitor NK cell localization and activation to better predict therapy responses, determine causes of therapy failure, and facilitate the clinical translation of emerging NK cell cancer therapies. To address this challenge, positron emission tomography (PET) tracers specific for NKp30 (NCR3) were developed and validated in vivo. NKp30 is expressed almost exclusively on NK cells along with rare ILC1 subtypes and highly upregulated in activated NK cells. PET imaging allows quantitative, serial, non-invasive imaging. A variety of PET radionuclides are used, depending on the targeting probes’ pharmacokinetics. An NKp30-specific antibody was radiolabeled with either copper-64 (64Cu, t1/2 = 12.7h) or zirconium-89 (89Zr, t1/2 = 78.4h) to determine which probe offers superior contrast. 64Cu and 89Zr allow in vivo imaging out to 48 hours and 7 days post-injection, respectively. The antibody was conjugated with the chelators DOTA or DFO followed by 64Cu or 89Zr radiolabeling. 64Cu-NKp30 had a radiochemical yield of 70-75% and specific activity of 7.5-10 uCi/ug, whereas 89Zr-NKp30 had a radiochemical yield of &amp;gt;85% and a specific activity of 4-5 uCi/ug. HPLC results demonstrated radiochemical purity of &amp;gt;98% and no antibody aggregation or fragmentation. To evaluate the binding specificity of these tracers in vivo, HeLa cells stably expressing NKp30 were subcutaneously implanted into flanks of nu/nu mice, along with HeLa wild-type (WT) cells in the opposite flank. 15 ug of 64Cu-NKp30 or 89Zr-NKp30 was injected, and PET/CT imaging completed at 24 and 48h for 64Cu-NKp30 and every 24h to 144h for 89Zr-NKp30. 64Cu-NKp30 had 14.4±4.4 %Id/g uptake in NKp30 xenografts, compared to 5.8±1.4 %Id/g for WT cells (p= 0.042, n=3) at 48h. 89Zr-NKp30 had 22.9±1.9 %Id/g uptake in NKp30 xenografts compared to 7.3±0.9 %Id/g for WT cells (p=0.0027, n=3) at 144h. Tumor/blood ratios were 1.6 for 64Cu-NKp30 vs 4.7 89Zr-NKp30 at 48 and 144 hours, respectively. These results demonstrate specific uptake in cells expressing NKp30 by both probes, with 89Zr-NKp30 at 144h demonstrating superior contrast. We have developed NKp30-specific PET tracers for imaging activated NK cell distribution with the potential for monitoring of NK cell immunotherapy in vivo. Citation Format: Travis M. Shaffer, Amin Aalipour, Sanjiv S. Gambhir. Positron emission tomography (PET) imaging of the natural killer (NK) cell activation receptor NKp30 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B43.