Time-course RNAseq data of murine AB1 mesothelioma and Renca renal cancer following immune checkpoint therapy.

Time-critical transcriptional events in the immune microenvironment are important for response to immune checkpoint blockade (ICB), yet these events are difficult to characterise and remain incompletely understood. Here, we present whole tumor RNA sequencing data in the context of treatment with ICB in murine models of AB1 mesothelioma and Renca renal cell cancer. We sequenced 144 bulk RNAseq samples from these two cancer types across 4 time points prior and after treatment with ICB. We also performed single-cell sequencing on 12 samples of AB1 and Renca tumors an hour before ICB administration. Our samples were equally distributed between responders and non-responders to treatment. Additionally, we sequenced AB1-HA mesothelioma tumors treated with two sample dissociation protocols to assess the impact of these protocols on the quality transcriptional information in our samples. These datasets provide time-course information to transcriptionally characterize the ICB response and provide detailed information at the single-cell level of the early tumor microenvironment prior to ICB therapy.

Early de-escalation of antibiotic therapy in hospitalized cellular therapy adult patients with febrile neutropenia.

Febrile neutropenia (FN) is an oncologic emergency frequently encountered in hematopoietic cell transplant (HCT) and chimeric antigen receptor (CAR) T-cell therapy patients, which requires immediate initiation of broad-spectrum antibiotics. Data regarding antibiotic de-escalation (DE) in neutropenic patients are limited, and guideline recommendations vary. A clinical protocol for antibiotic DE of broad-spectrum agents was implemented if patients were afebrile after 72 hours and had no clinical evidence of infection. The primary endpoint was the difference in the number of antibiotic therapy days between the pre-and post-DE protocol implementation group. Secondary endpoints included rates of subsequent bacteremia during index hospitalization, 30-day mortality, and hospital length of stay. Retrospective chart reviews were conducted to assess outcomes for patients who received allogeneic HCT, autologous HCT, or CAR T-cell therapy under the antibiotic de-escalation protocol (post-DE) compared to those who did not (pre-DE). The pre-DE group underwent HCT/CAR T-cell from February 2018 through September 2018 (n=64), and the post-DE group from February 2019 through September 2019 (n=67). The median duration of antibiotics was significantly lower in the post-DE group (6 days; range 3-60 days) compared to the pre-DE group (8 days; range 3-31 days) (p=0.034). There were no differences in any secondary endpoints. We conclude that antibiotic DE in neutropenic HCT or CAR T-cell therapy patients treated with broad-spectrum antibiotics for at least three days who are afebrile and without documented infection appears to be a safe and effective practice. Adopting it significantly reduces the number of days of antibiotics without compromising patient outcomes.

Immune checkpoint therapy responders display early clonal expansion of tumor infiltrating lymphocytes.

Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRß) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRß repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRß repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRß clonotypes was observed in responding tumours. Machine learning identified TCRß CDR3 signatures unique to each tumour model, and signatures associated with ICT response at various timepoints before or during ICT. Clonally expanded CD8+ T cells in responding tumours post ICT displayed effector T cell gene signatures and phenotype. An early burst of clonal expansion during ICT is associated with response, and we report unique dynamics in TCRß signatures associated with ICT response.

CRISPR-Cas9-generated PTCHD1 2489T>G stem cells recapitulate patient phenotype when undergoing neural induction.

An estimated 3.5%-5.9% of the global population live with rare diseases, and approximately 80% of these diseases have a genetic cause. Rare genetic diseases are difficult to diagnose, with some affected individuals experiencing diagnostic delays of 5-30 years. Next-generation sequencing has improved clinical diagnostic rates to 33%-48%. In a majority of cases, novel variants potentially causing the disease are discovered. These variants require functional validation in specialist laboratories, resulting in a diagnostic delay. In the interim, the finding is classified as a genetic variant of uncertain significance (VUS) and the affected individual remains undiagnosed. A VUS (PTCHD1 c. 2489T>G) was identified in a child with autistic behavior, global developmental delay, and hypotonia. Loss of function mutations in PTCHD1 are associated with autism spectrum disorder and intellectual disability; however, the molecular function of PTCHD1 and its role in neurodevelopmental disease is unknown. Here, we apply CRISPR gene editing and induced pluripotent stem cell (iPSC) neural disease modeling to assess the variant. During differentiation from iPSCs to neural progenitors, we detect subtle but significant gene signatures in synaptic transmission and muscle contraction pathways. Our work supports the causal link between the genetic variant and the child's phenotype, providing evidence for the variant to be considered a pathogenic variant according to the American College of Medical Genetics and Genomics guidelines. In addition, our study provides molecular data on the role of PTCHD1 in the context of other neurodevelopmental disorders.

Gene editing and cardiac disease modelling for the interpretation of genetic variants of uncertain significance in congenital heart disease.

BACKGROUND: Genomic sequencing in congenital heart disease (CHD) patients often discovers novel genetic variants, which are classified as variants of uncertain significance (VUS). Functional analysis of each VUS is required in specialised laboratories, to determine whether the VUS is disease causative or not, leading to lengthy diagnostic delays. We investigated stem cell cardiac disease modelling and transcriptomics for the purpose of genetic variant classification using a GATA4 (p.Arg283Cys) VUS in a patient with CHD. METHODS: We performed high efficiency CRISPR gene editing with homology directed repair in induced pluripotent stem cells (iPSCs), followed by rapid clonal selection with amplicon sequencing. Genetic variant and healthy matched control cells were compared using cardiomyocyte disease modelling and transcriptomics. RESULTS: Genetic variant and healthy cardiomyocytes similarly expressed Troponin T (cTNNT), and GATA4. Transcriptomics analysis of cardiomyocyte differentiation identified changes consistent with the patient's clinical human phenotype ontology terms. Further, transcriptomics revealed changes in calcium signalling, and cardiomyocyte adrenergic signalling in the variant cells. Functional testing demonstrated, altered action potentials in GATA4 genetic variant cardiomyocytes were consistent with patient cardiac abnormalities. CONCLUSIONS: This work provides in vivo functional studies supportive of a damaging effect on the gene or gene product. Furthermore, we demonstrate the utility of iPSCs, CRISPR gene editing and cardiac disease modelling for genetic variant interpretation. The method can readily be applied to other genetic variants in GATA4 or other genes in cardiac disease, providing a centralised assessment pathway for patient genetic variant interpretation.

A surgically optimized intraoperative poly(I:C)-releasing hydrogel prevents cancer recurrence.

Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects. Here, we develop a surgically optimized biodegradable hyaluronic acid-based hydrogel for sustained intraoperative delivery of Toll-like receptor 3 agonist poly(I:C) and demonstrate that it significantly reduces tumor recurrence after surgery in multiple mouse models. Mechanistically, poly(I:C) induces a transient interferon alpha (IFNα) response, reshaping the tumor/wound microenvironment by attracting inflammatory monocytes and depleting regulatory T cells. We demonstrate that a pre-existing IFN signature predicts response to the poly(I:C) hydrogel, which sensitizes tumors to immune checkpoint therapy. The safety, immunogenicity, and surgical feasibility are confirmed in a veterinary trial in canine soft tissue tumors. The surgically optimized poly(I:C)-loaded hydrogel provides a safe and effective approach to prevent cancer recurrence.

CD4+ T cells drive an inflammatory, TNF-α/IFN-rich tumor microenvironment responsive to chemotherapy.

While chemotherapy remains the first-line treatment for many cancers, it is still unclear what distinguishes responders from non-responders. Here, we characterize the chemotherapy-responsive tumor microenvironment in mice, using RNA sequencing on tumors before and after cyclophosphamide, and compare the gene expression profiles of responders with progressors. Responsive tumors have an inflammatory and highly immune infiltrated pre-treatment tumor microenvironment characterized by the enrichment of pathways associated with CD4+ T cells, interferons (IFNs), and tumor necrosis factor alpha (TNF-α). The same gene expression profile is associated with response to cyclophosphamide-based chemotherapy in patients with breast cancer. Finally, we demonstrate that tumors can be sensitized to cyclophosphamide and 5-FU chemotherapy by pre-treatment with recombinant TNF-α, IFNγ, and poly(I:C). Thus, a CD4+ T cell-inflamed pre-treatment tumor microenvironment is necessary for response to chemotherapy, and this state can be therapeutically attained by targeted immunotherapy.

Preliminary Factor Structure of the Parental Attitudes of Gender Expansiveness Scale for Parents (PAGES-P).

Objective: Parental acceptance and support are associated with positive psychosocial outcomes among transgender and gender expansive (TGE) adolescents. Understanding the degree of parental acceptance and support of gender identity and expression is an important component of gender affirmative pediatric assessment and can inform intervention. Although there are reliable measures assessing general family support, there are no existing parent self-report measures assessing acceptance and support of their gender expansive children. The present study examines the factor structure of the Parental Attitudes of Gender Expansiveness Scale for Parents (PAGES-P). Methods: Participants included 739 parents who completed the PAGES-P as standard-of-care during their child's gender health clinic visit within a children's hospital in the Midwestern United States. Principal Component Analysis (PCA) was used to identify subscales reflected in the PAGES-P. Results: PCA yielded four subscales reflecting the following domains: (1) support and affirmation, (2) guilt and loss, (3) gender concealment, and (4) pride. Conclusions: This study provides preliminary evidence of the factor structure of the PAGES-P. The resulting subscales lend insight into the thoughts and behaviors of parents of TGE youth and can inform clinical practice to facilitate parental support and promote overall well-being in TGE youth.

Functional validation of variants of unknown significance using CRISPR gene editing and transcriptomics: A Kleefstra syndrome case study.

There are an estimated > 400 million people living with a rare disease globally, with genetic variants the cause of approximately 80% of cases. Next Generation Sequencing (NGS) rapidly identifies genetic variants however they are often of unknown significance. Low throughput functional validation in specialist laboratories is the current ad hoc approach for functional validation of genetic variants, which creating major bottlenecks in patient diagnosis. This study investigates the application of CRISPR gene editing followed by genome wide transcriptomic profiling to facilitate patient diagnosis. As proof-of-concept, we introduced a variant in the Euchromatin histone methyl transferase (EHMT1) gene into HEK293T cells. We identified changes in the regulation of the cell cycle, neural gene expression and suppression of gene expression changes on chromosome 19 and chromosome X, that are in keeping with Kleefstra syndrome clinical phenotype and/or provide insight into disease mechanism. This study demonstrates the utility of genome editing followed by functional readouts to rapidly and systematically validating the function of variants of unknown significance in patients suffering from rare diseases.

CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency.

BACKGROUND: Over 400 million people worldwide are living with a rare disease. Next Generation Sequencing (NGS) identifies potential disease causative genetic variants. However, many are identified as variants of uncertain significance (VUS) and require functional laboratory validation to determine pathogenicity, and this creates major diagnostic delays. METHODS: In this study we test a rapid genetic variant assessment pipeline using CRISPR homology directed repair to introduce single nucleotide variants into inducible pluripotent stem cells (iPSCs), followed by neuronal disease modelling, and functional genomics on amplicon and RNA sequencing, to determine cellular changes to support patient diagnosis and identify disease mechanism. RESULTS: As proof-of-principle, we investigated an EHMT1 (Euchromatin histone methyltransferase 1; EHMT1 c.3430C > T; p.Gln1144*) genetic variant pathogenic for Kleefstra syndrome and determined changes in gene expression during neuronal progenitor cell differentiation. This pipeline rapidly identified Kleefstra syndrome in genetic variant cells compared to healthy cells, and revealed novel findings potentially implicating the key transcription factors REST and SP1 in disease pathogenesis. CONCLUSION: The study pipeline is a rapid, robust method for genetic variant assessment that will support rare diseases patient diagnosis. The results also provide valuable information on genome wide perturbations key to disease mechanism that can be targeted for drug treatments.

Tumour draining lymph node-generated CD8 T cells play a role in controlling lung metastases after a primary tumour is removed but not when adjuvant immunotherapy is used.

Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting. In a murine metastatic lung cancer model, primary subcutaneous tumours were resected with associated dLNs remaining intact, completely resected or partially resected. Median survival after surgery was significantly shorter with complete dLN resection at the time of surgery (49 days (95%CI)) compared to when lymph nodes remained intact (> 88 days; p < 0.05). Survival was partially restored with incomplete lymph node resection and CD8 T cell dependent. Treatment with aCTLA4 whilst effective against the primary tumour was ineffective for metastatic lung disease. Conversely, aPD-1/aCD40 treatment was effective in both the primary and metastatic disease settings and restored the detrimental effects of complete dLN resection on survival. In this pre-clinical lung metastatic disease model that closely reflects the clinical setting, we observe decreased frequency of survival after complete lymphadenectomy, which was ameliorated with partial lymph node removal or with early administration of aPD-1/aCD40 therapy. These findings have direct relevance to surgical lymph node resection and adjuvant immunotherapy in lung cancer, and perhaps other cancer, patients.

Tumor Infiltrating Effector Memory Antigen-Specific CD8+ T Cells Predict Response to Immune Checkpoint Therapy.

Immune checkpoint therapy (ICT) results in durable responses in individuals with some cancers, but not all patients respond to treatment. ICT improves CD8+ cytotoxic T lymphocyte (CTL) function, but changes in tumor antigen-specific CTLs post-ICT that correlate with successful responses have not been well characterized. Here, we studied murine tumor models with dichotomous responses to ICT. We tracked tumor antigen-specific CTL frequencies and phenotype before and after ICT in responding and non-responding animals. Tumor antigen-specific CTLs increased within tumor and draining lymph nodes after ICT, and exhibited an effector memory-like phenotype, expressing IL-7R (CD127), KLRG1, T-bet, and granzyme B. Responding tumors exhibited higher infiltration of effector memory tumor antigen-specific CTLs, but lower frequencies of regulatory T cells compared to non-responders. Tumor antigen-specific CTLs persisted in responding animals and formed memory responses against tumor antigens. Our results suggest that increased effector memory tumor antigen-specific CTLs, in the presence of reduced immunosuppression within tumors is part of a successful ICT response. Temporal and nuanced analysis of T cell subsets provides a potential new source of immune based biomarkers for response to ICT.

Pre-treatment tumor neo-antigen responses in draining lymph nodes are infrequent but predict checkpoint blockade therapy outcome.

Immune checkpoint blockade (ICPB) is a powerfully effective cancer therapy in some patients. Tumor neo-antigens are likely main targets for attack but it is not clear which and how many tumor mutations in individual cancers are actually antigenic, with or without ICPB therapy and their role as neo-antigen vaccines or as predictors of ICPB responses. To examine this, we interrogated the immune response to tumor neo-antigens in a murine model in which the tumor is induced by a natural human carcinogen (i.e. asbestos) and mimics its human counterpart (i.e. mesothelioma). We identified and screened 33 candidate neo-antigens, and found T cell responses against one candidate in tumor-bearing animals, mutant UQCRC2. Interestingly, we found a high degree of inter-animal variation in the magnitude of neo-antigen responses in otherwise identical mice. ICPB therapy with Cytotoxic T-lymphocyte-associated protein (CTLA-4) and α-glucocorticoid-induced TNFR family related gene (GITR) in doses that induced tumor regression, increased the magnitude of responses and unmasked functional T cell responses against another neo-antigen, UNC45a. Importantly, the magnitude of the pre-treatment draining lymph node (dLN) response to UNC45a closely corresponded to ICPB therapy outcomes. Surprisingly however, boosting pre-treatment UNC45a-specific T cell numbers did not improve response rates to ICPB. These observations suggest a novel biomarker approach to the clinical prediction of ICPB response and have important implications for the development of neo-antigen vaccines.

Impaired T cell proliferation by ex vivo BET-inhibition impedes adoptive immunotherapy in a murine melanoma model.

Activation of naïve CD8+ T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of ex vivo activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties. Ex vivo targeting of T cell differentiation with epigenetic modifiers is a potential strategy to improve cytotoxic T-lymphocyte (CTL) generation for ACT. We established a pipeline to assess the effects of epigenetic modifiers on CD8+ T cell proliferation, differentiation, and efficacy in a preclinical melanoma model. Single treatment with epigenetic modifiers inhibited T cell proliferation in vitro, producing CD44hiCD62Lhi effector-like T cells rather than a stem cell memory T cell phenotype. Most epigenetic modifying agents had no significant effect on ACT efficacy with the notable exception of the bromodomain and extraterminal (BET)-inhibitor JQ1 which was associated with a decrease in efficacy compared to unmodified T cells. These findings reveal the complexity of epigenetic targeting of T cell differentiation, highlighting the need to precisely define the epigenetic targeting strategies to improve CTL generation for ACT.

Acquired resistance during adoptive cell therapy by transcriptional silencing of immunogenic antigens.

Immunotherapies such as adoptive cell therapy (ACT) are promising treatments for solid cancers. However, relapsing disease remains a problem and the molecular mechanisms underlying resistance are poorly defined. We postulated that the deregulated epigenetic landscape in cancer cells could underpin the acquisition of resistance to immunotherapy. To address this question, two preclinical models of ACT were employed to study transcriptional and epigenetic regulatory processes within ACT-treated cancer cells. In these models ACT consistently causes robust tumor regression, but resistance develops and tumors relapse. We identified down-regulated expression of immunogenic antigens at the mRNA level correlated with escape from immune control. To determine whether this down-regulation was under epigenetic control, we treated escaped tumor cells with DNA demethylating agents, azacytidine (AZA) and decitabine (DEC). AZA or DEC treatment restored antigen expression in a proportion of the tumor population. To explore the importance of other epigenetic modifications we isolated tumor cells refractory to DNA demethylation and screened clones against a panel of 19 different epigenetic modifying agents (EMAs). The library of EMAs included inhibitors of a range of chromosomal and transcription regulatory protein complexes, however, when tested as single agents none restored further antigen expression. These findings suggest that tumor cells employ multiple epigenetic and genetic mechanisms to evade immune control, and a combinatorial approach employing several EMAs targeting transcription and genome stability may be required to overcome tumor resistance to immunotherapy.

Combination immune checkpoint blockade as an effective therapy for mesothelioma.

Mesothelioma is an aggressive asbestos induced cancer with extremely poor prognosis and limited treatment options. Immune checkpoint blockade (ICPB) has demonstrated effective therapy in melanoma and is now being applied to other cancers, including mesothelioma. However, the efficacy of ICPB and which immune checkpoint combinations constitute the best therapeutic option for mesothelioma have yet to be fully elucidated. Here, we used our well characterised mesothelioma tumour model to investigate the efficacy of different ICBP treatments to generate effective therapy for mesothelioma. We show that tumour resident regulatory T cell co-express high levels of CTLA-4, OX40 and GITR relative to T effector subsets and that these receptors are co-expressed on a large proportion of cells. Targeting any of CTLA-4, OX40 or GITR individually generated effective responses against mesothelioma. Furthermore, the combination of αCTLA-4 and αOX40 was synergistic, with an increase in complete tumour regressions from 20% to 80%. Other combinations did not synergise to enhance treatment outcomes. Finally, an early pattern in T cell response was predictive of response, with activation status and ICP receptor expression profile of T effector cells harvested from tumour and dLN correlating with response to immunotherapy. Taken together, these data demonstrate that combination ICPB can work synergistically to induce strong, durable immunity against mesothelioma in an animal model.

Perceived Parental Attitudes of Gender Expansiveness: Development and Preliminary Factor Structure of a Self-Report Youth Questionnaire.

Purpose: Parental acceptance of gender identity/expression in lesbian, gay, bisexual, transgender, and queer/questioning (LGBTQ+) youth moderates the effects of minority stress on mental health outcomes. Given this association, mental health clinicians of gender-expansive adolescents often assess the degree to which these youth perceive their parents/primary caregivers as accepting or nonaffirming of their gender identity and expression. While existing measures may reliably assess youth's perceptions of general family support, no known tool aids in the assessment an adolescent's perceived parental support related to adolescent gender-expansive experiences. Methods: To provide both clinicians and researchers with an empirically derived tool, the current study used factor analysis to explore an underlying factor structure of a brief questionnaire developed by subject-matter experts and pertaining to multiple aspects of perceived parental support in gender-expansive adolescents and young adults. Respondents were gender-expansive adolescents and young adults seeking care in an interdisciplinary gender-health clinic within a pediatric academic medical center in the Midwestern United States. Results: Exploratory factor analysis resulted in a 14-item questionnaire comprised of two subscales assessing perceived parental nonaffirmation and perceived parental acceptance. Internal consistency and construct validity results provided support for this new questionnaire. Conclusion: This study provides preliminary evidence of the factor structure, reliability and validity of the Parental Attitudes of Gender Expansiveness Scale for Youth (PAGES-Y). These findings demonstrate both the clinical and research utility of the PAGES-Y, a tool that can yield a more nuanced understanding of family-related risk and protective factors in gender-expansive adolescents.

Ly49C Impairs NK Cell Memory in Mouse Cytomegalovirus Infection.

NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function, accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed "licensing." Ly49C is the main self-specific inhibitory Ly49 receptor in H-2(b) C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 ß2 microglobulin (ß2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells. We found that self-specific inhibitory receptors affected NK cell precursor survival and proliferation at particular developmental stages in an MHC class I-dependent manner. The presence of Ly49C impacted the NK cell repertoire in a ß2m-dependent manner, with reduced Ly49A(+), Ly49G2(+), and Ly49D(+) subsets, an increased DNAM-1(+) subset, and higher NKG2D expression. Licensed NK cells displayed a skewed distribution of the maturation stages, which was characterized by differential CD27 and CD11b expression, toward the mature phenotypes. We found that Ly49C-mediated licensing induced a split effect on NK cell functions, with increased cytokine-production capabilities following engagement of various activating receptors while cytotoxicity remained unchanged. Analysis of licensed NK cell functions in vivo, in a system of mouse CMV infection, indicated that licensing did not play a major role in the NK cell antiviral response during acute infection, but it strongly impaired the generation and/or persistence of memory NK cells. This study unravels multifaceted effects of licensing on NK cell populations and their functions.

Ly49C-dependent control of MCMV Infection by NK cells is cis-regulated by MHC Class I molecules.

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMVG1F) whose variant m157 also binds the inhibitory Ly49C receptor. Here we show that simultaneous binding of m157 to the two receptors hampers Ly49H-dependent NK cell activation as Ly49C-mediated inhibition destabilizes NK cell conjugation with their targets and prevents the cytoskeleton reorganization that precedes killing. In B6 mice, as most Ly49H+ NK cells do not co-express Ly49C, the overall NK cell response remains able to control MCMVm157G1F infection. However, in B6 Ly49C transgenic mice where all NK cells express the inhibitory receptor, MCMV infection results in altered NK cell activation associated with increased viral replication. Ly49C-mediated inhibition also regulates Ly49H-independent NK cell activation. Most interestingly, MHC class I regulates Ly49C function through cis-interactions that mask the receptor and restricts m157 binding. B6 Ly49C Tg, ß2m ko mice, whose Ly49C receptors are unmasked due to MHC class I deficient expression, are highly susceptible to MCMVm157G1F and are unable to control a low-dose infection. Our study provides novel insights into the mechanisms that regulate NK cell activation during viral infection.