The current landscape of systemic therapy for recurrent glioblastoma: A systematic review of randomized-controlled trials.

AIM: Conduct a systematic review of the effectiveness of systemic therapies for adult recurrent glioblastoma (rGBM). METHODS: We electronically searched for randomized controlled trials from three major databases and four conferences from 2009-Dec 2020. Two independent reviewers conducted screening, data extraction, and quality assessment. RESULTS: 48 randomized trials were identified. Outcome reporting was inconsistent: overall survival (OS) in 46 studies, progression free survival in 37 studies, 6-month PFS in 30 studies, objective response rate in 28 studies, and 6-month OS in 7 studies. Network meta-analysis was not feasible due to heterogeneity in outcome reporting and single-study linkages. Most studies compared lomustine (8 studies), bevacizumab (18), or temozolomide (8) with other treatments. The median OS across all studies ranged from 3 to 17.6 months. CONCLUSIONS: Based on level one evidence, there is no superior systemic regimen for rGBM. rGBM is a heterogeneous population with no single regimen demonstrating OS benefit. Registration number: CRD42020148512.

Two-year results of a randomised trial comparing 4- versus 12-weekly bone-targeted agent use in patients with bone metastases from breast or castration-resistant prostate cancer.

BACKGROUND: We present the 2-year results of a randomised trial comparing 4- versus 12-weekly bone-targeting agents (BTAs) in patients with bone metastases from breast or castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: Patients with bone metastases from breast or CRPC, who were going to start or were already receiving BTAs, were randomised to 4- or 12-weekly BTA treatment for 2 years. The endpoints were: symptomatic skeletal events (SSE) rates, time to SSEs, toxicity and cost-effectiveness. RESULTS: Of 263 patients (160 breast cancer, 103 CRPC), 133 (50.6%) and 130 (49.4%) were randomised to the 4- and 12-weekly groups, respectively. BTAs included denosumab (56.3%), zoledronate (24.0%) and pamidronate (19.8%). After 2 years, the cumulative incidence rate (95% CI) of SSEs was 32.7% (24.6% to 41.1%) and 28.1% (20.3% to 36.4%) for the 4- and 12-weekly intervention groups respectively. The hazard ratio for time to first SSE was 0.96 (95% CI = 0.63 to 1.47). However, in a post hoc analysis, those patients who had an on-study SSE, there was a small non-statistical increased risk of subsequent SSEs among patients on the 12-weekly dosing arm (HR = 1.14; 95% CI - 0.90-1.44). BTA-related toxicity rates were similar between study arms. A cost-utility analysis showed that 12-weekly BTA is cost-effective from a public payer's perspective. CONCLUSION: These results in addition to those previously reported for de-escalating zoledronate, would support that de-escalation of commonly used BTAs is a reasonable and economically valid treatment option. While not statistically significant, the increase in subsequent SSEs in the 12-weekly arm requires further exploration.

Cost-Effectiveness Analysis of 12-Versus 4-Weekly Administration of Bone-Targeted Agents in Patients with Bone Metastases from Breast and Castration-Resistant Prostate Cancer.

A cost-utility analysis was performed based on the Rethinking Clinical Trials (REaCT) bone-targeted agents (BTA) clinical trial that compared 12-weekly (once every 12 weeks) (n = 130) versus 4-weekly (once every 4 weeks) (n = 133) BTA dosing for metastatic breast and castration-resistant prostate (CRPC) cancer. Using a decision tree model, we calculated treatment and symptomatic skeletal event (SSE) costs as well as quality-adjusted life-years (QALYs) for each treatment option. Deterministic and probabilistic sensitivity analyses were performed to assess the robustness of the study findings. The total cost of BTA treatment in Canadian dollars (C$) and estimated QALYs was C$8965.03 and 0.605 QALY in the 4-weekly group versus C$5669.95 and 0.612 QALY in the 12-weekly group, respectively. De-escalation from 4-weekly to 12-weekly BTA reduces cost (C$3293.75) and improves QALYs by 0.008 unit, suggesting that 12-weekly BTA dominates 4-weekly BTA in breast and CRPC patients with bone metastases. Sensitivity analysis suggests high levels of uncertainty in the cost-effectiveness findings. De-escalation of bone-targeted agents is cost-effective from the Canadian public payer's perspective.

Long-term impact of bone-modifying agents for the treatment of bone metastases: a systematic review.

PURPOSE: Bone-modifying agents (BMAs) for bone metastases are commonly prescribed for many years even though randomized clinical trials are only 1-2 years in duration. A systematic review on the risk-benefit of BMA use for > 2 years in breast cancer or castrate-resistant prostate cancer was conducted. METHODS: MEDLINE, Embase, and Cochrane databases were searched (1970-February 2019) for randomized and observational studies, and case series reporting on BMA efficacy (skeletal-related events and quality of life) and toxicity (osteonecrosis of the jaw, renal impairment, hypocalcemia, and atypical femoral fractures) beyond 2 years. RESULTS: Of 2107 citations, 64 studies were identified. Three prospective and 9 retrospective studies were eligible. Data beyond 2 years was limited to subgroup analyses in all studies. Only one study (n = 181) reported skeletal-related event rates based on bisphosphonate exposure, with decreased rates from 27.6% (0-24 months) to 15.5% (> 24 months). None reported on quality of life. All 12 studies (denosumab (n = 948), zoledronate (n = 1036), pamidronate (n = 163), pamidronate-zoledronate (n = 522), ibandronate (n = 118)) reported ≥ 1 toxicity outcome. Seven bisphosphonate studies (n = 1077) and one denosumab study (n = 948) reported on osteonecrosis of the jaw. Across three studies (n = 1236), osteonecrosis of the jaw incidence ranged from 1 to 4% in the first 2 years to 3.8-18% after 2 years. Clinically significant hypocalcemia ranged from 1 to 2%. Severe renal function decline was ≤ 3%. Atypical femoral fractures were rare. CONCLUSIONS: Evidence informing the use of BMA beyond 2 years is heterogeneous and based on retrospective analysis. Prospective randomized studies with greater emphasis on quality of life are needed. PROSPERO REGISTRATION NUMBER: CRD42019126813.

Inhibition of the CCL2 receptor, CCR2, enhances tumor response to immune checkpoint therapy.

Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8+ T cell recruitment and activation and a concomitant decrease in CD4+ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies.

Targeting DDR2 enhances tumor response to anti-PD-1 immunotherapy.

While a fraction of cancer patients treated with anti-PD-1 show durable therapeutic responses, most remain unresponsive, highlighting the need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies-bladder, breast, colon, sarcoma, and melanoma-we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, led to tumor load reduction. RNA-seq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors.

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.

ROS1 Gene Rearrangements Are Associated With an Elevated Risk of Peridiagnosis Thromboembolic Events.

INTRODUCTION: This study aims to determine whether advanced ROS1 gene-rearranged NSCLC (ROS1+ NSCLC) has a higher than expected thromboembolic event (TEE) rate. METHODS: Venous and arterial TEEs within ±365 days of diagnosis of ROS1+, ALK+, EGFR+, or KRAS+ advanced NSCLC at five academic centers in the United States and China were captured (October 2002-April 2018). The primary endpoint was incidence of TEE in ROS1+ compared to anaplastic lymphoma kinase (ALK)+, EGFR+, and KRAS+ NSCLC within ±90 days of diagnosis. Logistic regression was used to assess if the odds of TEE differed among oncogene drivers. RESULTS: Eligible data from 95 ROS1+, 193 ALK+, 300 EGFR+, and 152 KRAS+ NSCLC patients were analyzed. The incidence rate of TEE was 34.7% (n = 33), 22.3% (n = 43), 13.7% (n = 41), and 18.4% (n = 28), respectively. In univariate analysis, the odds of a TEE in ROS1+ NSCLC were higher than ALK+, EGFR+, and KRAS+ cohorts. In multivariable analysis, the odds of a TEE were significantly higher for ROS1+ compared to EGFR+ and KRAS+ cohorts, the odds ratio (OR) was 2.44, with a 95% confidence interval of 1.31-4.57 (p = 0.005), and OR: 2.62, with a 95% confidence interval of 1.26-5.46 (p = 0.01), respectively. Although numerically superior, the odds for a TEE with ROS1+ compared to ALK+ was not statistically significant (OR: 1.45, p = 0.229). Overall survival was not significantly different in patients with or without TEE within ±90 days of diagnosis in the overall study cohort or within each molecular group. CONCLUSIONS: The risk of peridiagnostic TEEs is significantly elevated in patients with advanced ROS+ NSCLC compared to EGFR+ and KRAS+ cases. TEE risk may be similarly elevated in ALK+ NSCLC.

Molecular Biomarkers of Response to PD-1/ PD-L1 Immune Checkpoint Blockade in Advanced Bladder Cancer.

BACKGROUND: The activity of PD-1/PD-L1 inhibitors in the treatment of advanced bladder cancer (BC) is promising for many patients. However, a subset of patients do not benefit from treatment, thus leading to an effort to better identify predictive molecular biomarkers of response. OBJECTIVE: To conduct a systematic review of the literature on predictive molecular biomarkers associated with response to PD-1 and PD-L1 inhibitors in advanced bladder cancer, defined as locally-advanced, unresectable, or metastatic (mBC) disease. METHODS: A search of the literature was performed using Embase (1947 - January 2019), Medline (1946 - January 2019), and EBM Reviews for Cochrane Central Register of Controlled Trials (as of December 2018). Studies examining the association of molecular biomarkers with clinical outcome in BC treated with PD-1 or PD-L1 monotherapy were included. Outcomes of interest were overall survival (OS), cancer-specific survival (CSS), progression-free survival (PFS), duration of response, and objective response rate (ORR). RESULTS: Using the study search criteria, 899 unique abstract citations were found, of which 834 did not meet the eligibility criteria. Full text of the remaining 65 citations were screened, and 50 studies excluded, including 18 review articles. Eight additional studies from the bibliography of the review papers were included, making a total of 23 studies. Five PD-1 / PD-L1 antibodies have been tested in BC immunohistochemistry (IHC). These studies used different expression scoring criteria and generally had poor ability to discriminate likelihood for response. Overall, the data suggests CD8+ T cell infiltration is necessary to mediate an antitumor immune response, but other immune cell populations, such as neutrophils may suppress T cell-mediated immunity and efficacy of PD-1/PD-L1 blockade. An IFNγ signature is a promising predictor, but there needs to be consensus on the optimal gene panel composition, and prospective validation. Tumor mutation burden (TMB) is a promising predictor in six studies reporting on 1200 patients, but there is not a consensus on the optimal definition of "high TMB". Detection of T cell receptor (TCR) clonal expansion has only been conducted in small studies and so its predictive value remains inconclusive. Epithelial-mesenchymal transformation (EMT) and transforming growth factor ß (TGFß) are associated with poor prognosis and possibly intrinsic resistance to PD-1/PD-L1 checkpoint blockade, but more work needs to be done to build upon and confirm the initial findings. CONCLUSIONS: Currently no molecular biomarker is sufficiently mature for routine clinical use, while some candidates, or a combination show great promise and need further study.

The immune checkpoint, HVEM may contribute to immune escape in non-small cell lung cancer lacking PD-L1 expression.

BACKGROUND: Herpes Virus Entry Mediator (HVEM) is an important immune checkpoint in cancer recognition. HVEM expressed on tumor cell membranes activates immune cell signaling pathways leading to either inhibition of activity (B- and T-lymphocyte attenuator, BTLA) or activation of immune activity (LIGHT). The aim of this study is to investigate the prevalence of HVEM expression and its association with PDL1 expression in NSCLC. METHODS: A TMA of 527 resected NSCLC samples and 56 NSCLC cell lines were evaluated for HVEM and PD-L1 expression. The IHC assay for HVEM was optimized on the Dako Link48 autostainer using a polyclonal antibody from R&D Systems(AF356). PD-L1 IHC was performed on the Dako Link48 autostainer using the PD-L1 28-8 pharmDx kit. Scoring HVEM employed the H-score system while for PD-L1 the tumor proportion score (TPS) was used. RESULTS: HVEM expression in the NSCLC resected samples and cell lines revealed a positive H-score more than 1 was 18.6% (77/415) and 48.2% (27/56) respectively. HVEM expression was significantly higher in patients with lymph node N2 metastasis (25.5% vs 7.9% vs 17.5%, p = 0.046) when comparing with N1 or no lymph node metastasis, and was marginally significantly higher in patients with stage III/IV disease (24.5% vs 16.4%, p = 0.059). Subgroup analysis showed that HVEM (median 45 vs 36 months, p = 0.706) and PD-L1 expression (median 45 vs 48 months, p = 0.178) status was not predictive of overall survival. HVEM was found to have a significant negative correlation with PD-L1 expression (r=-0.274, p < 0.001) in patients with NSCLC and also a weak negative correlation in NSCLC cell lines (r=-0.162, p = 0.352). CONCLUSION: HVEM was found to be overexpressed in NSCLC patients of N2 lymph node metastasis or later stage and has a negative co-relationship with PD-L1 expression. HVEM was not prognostic for NSCLC patients.

Critical role for the Ly49 family of class I MHC receptors in adaptive natural killer cell responses.

Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.

Immunosurveillance and Immunoediting of Breast Cancer via Class I MHC Receptors.

Ly49 receptors, which recognize "self" class I major histocompatibility complex (MHC-I) molecules, enable natural killer (NK) cells to detect loss of MHC-I expression on transformed and virally infected cells. The impact of NK cell-mediated MHC-I surveillance on immunoediting of breast cancer is still not fully understood. This work assesses the impact of Ly49 receptors on tumor development in terms of cancer control and in driving immune-evading cancer mutations. Genetically modified Ly49-deficient mice and those lacking NK cells through antibody depletion were less able to control E0771-derived mammary tumors in an MHC-I-dependent fashion. Similarly, Ly49-deficient MMTV-PyVT-transgenic mice developed spontaneous mammary tumors faster than Ly49-sufficient MMTV-PyVT mice. Fewer CD69+ and granzyme B+ NK cells were detected among the tumor-infiltrating lymphocytes in Ly49-deficient than in Ly49-sufficient MMTV-PyVT mice. Furthermore, tumors from Ly49-deficient mice displayed reduced MHC-I expression, suggesting that tumors growing in these mice lacked an Ly49-derived pressure to maintain MHC-I expression. These same MHC-I-low tumors from Ly49-deficient mice were unable to flourish when transferred to Ly49-sufficient hosts, confirming that this tumor mutation was in response to an Ly49-deficient environment. This work demonstrates a role for Ly49 receptors in the control of mammary cancer, and provides evidence to support a model of tumor immunoediting, in which selective pressures from the immune system drive immune-evasive cancer mutations. Cancer Immunol Res; 5(11); 1016-28. ©2017 AACR.

Licensed and Unlicensed NK Cells: Differential Roles in Cancer and Viral Control.

Natural killer (NK) cells are known for their well characterized ability to control viral infections and eliminate tumor cells. Through their repertoire of activating and inhibitory receptors, NK cells are able to survey different potential target cells for various surface markers, such as MHC-I - which signals to the NK cell that the target is healthy - as well as stress ligands or viral proteins, which alert the NK cell to the aberrant state of the target and initiate a response. According to the "licensing" hypothesis, interactions between self-specific MHC-I receptors - Ly49 in mice and KIR in humans - and self-MHC-I molecules during NK cell development is crucial for NK cell functionality. However, there also exists a large proportion of NK cells in mice and humans, which lack self-specific MHC-I receptors and are consequentially "unlicensed." While the licensed NK cell subset plays a major role in the control of MHC-I-deficient tumors, this review will go on to highlight the important role of the unlicensed NK cell subset in the control of MHC-I-expressing tumors, as well as in viral control. Unlike the licensed NK cells, unlicensed NK cells seem to benefit from the lack of self-specific inhibitory receptors, which could otherwise be exploited by some aberrant cells for immunoevasion by upregulating the expression of ligands or mimic ligands for these receptors.

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

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.

Ly49 receptors activate angiogenic mouse DBA⁺ uterine natural killer cells.

In humans, specific patterns of killer immunoglobulin-like receptors (KIRs) expressed by uterine natural killer (uNK) cells are linked through HLA-C with pregnancy complications (infertility, recurrent spontaneous abortion, intrauterine growth restriction and preeclampsia). To identify mechanisms underpinning the associations between NK cell activation and pregnancy success, pregnancies were studied in mice with genetic knockdown (KD) of the MHC-activated Ly49 receptor gene family. B6.Ly49(KD) pregnancies were compared to normal control B6.Ly49(129) and C57BL/6 (B6) pregnancies. At mid-pregnancy (gestation day (gd9.5)), overall uNK cell (TCRß(-)CD122(+)DBA(+)DX5(-) (DBA(+)DX5(-))) and TCRß(-)CD122(+)DBA(-)DX5(+) (DBA(-)DX5(+))) frequencies in pregnant uterus were similar between genotypes. Ly49(KD) lowered the normal frequencies of Ly49(+) uNK cells from 90.3% to 47.8% in DBA(-)DX5(+) and 78.8% to 6.3% in DBA(+)DX5(-) uNK cell subtypes. B6.Ly49(KD) matings frequently resulted in expanded blastocysts that did not implant (subfertility). B6.Ly49(KD) mice that established pregnancy had gestational lengths and litter sizes similar to controls. B6.Ly49(KD) neonates, however, were heavier than controls. B6.Ly49(KD) implantation sites lagged in early (gd6.5) decidual angiogenesis and were deficient in mid-pregnancy (gd10.5) spiral arterial remodelling. Ultrastructural analyses revealed that B6.Ly49(KD) uNK cells had impaired granulogenesis, while immunocytochemistry revealed deficient vascular endothelial cell growth factor (VEGFA) production. Perforin and IFNG expression were normal in B6.Ly49(KD) uNK cells. Thus, in normal mouse pregnancies, Ly49 receptor signaling must promote implantation, early decidual angiogenesis and mid-pregnancy vascular remodelling. Disturbances in these functions may underlie the reported genetic associations between human pregnancy complications and the inability of specific conceptus MHCs to engage activating KIR on uNK cells.

Ly49 family receptors are required for cancer immunosurveillance mediated by natural killer cells.

According to the missing-self hypothesis, natural killer (NK) cells survey for target cells that lack MHC-I molecules. The Ly49 receptor family recognizes loss of MHC-I and is critical for educating NK cells, conferring the ability to eliminate transformed or infected cells. In this study, we evaluated their requirement in innate immune surveillance of cancer cells using genetically manipulated mice with attenuated expression of Ly49 receptors (NKC(KD)) in several models of carcinoma and metastasis. We found that NKC(KD) mice exhibited uncontrolled tumor growth and metastases. Expression of two MHC-I alleles, H-2K(b) and H-2D(b), was decreased in tumors from NKC(KD) mice in support of the likelihood of NK-mediated tumor immunoediting. These tumor cells exhibited directed alterations to their cell surface expression in response to the genetically altered immune environment to evade host recognition. Immunoediting in NKC(KD) mice was restricted to MHC-I molecules, which are ligands for Ly49 receptors, while expression of Rae-1 and Mult1, ligands for another NK cell receptor, NKG2D, were unaffected. Restoring NK cell education in NKC(KD) mice with a transgene for the inhibitory self-MHC-I receptor Ly49I restored suppression of cancer onset and growth. Interestingly, immune surveillance mediated by activating Ly49 receptors remained intact in NKC(KD) mice, as demonstrated by the ability to stimulate the NKG2D receptor with tumor cells or splenocytes expressing Rae-1. Together, our results genetically establish the integral role of Ly49 in NK cell-mediated control of carcinogenesis through MHC-I-dependent missing-self recognition.

Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.