Time-dependent regulation of cytokine production by RNA binding proteins defines T cell effector function.

Potent T cell responses against infections and malignancies require a rapid yet tightly regulated production of toxic effector molecules. Their production level is defined by post-transcriptional events at 3' untranslated regions (3' UTRs). RNA binding proteins (RBPs) are key regulators in this process. With an RNA aptamer-based capture assay, we identify >130 RBPs interacting with IFNG, TNF, and IL2 3' UTRs in human T cells. RBP-RNA interactions show plasticity upon T cell activation. Furthermore, we uncover the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine production, ZFP36L1, ATXN2L, and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion does not rescue the dysfunctional phenotype, tumor-infiltrating T cells produce more cytokines and cytotoxic molecules, resulting in superior anti-tumoral T cell responses. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease.

WNT10A and RUNX2 mutations associated with non-syndromic tooth agenesis.

The goal of this study was to examine the prevalence of WNT10A and RUNX2 mutations and assess their potential impact on the phenotype of non-syndromic tooth agenesis. The study included 30 participants with non-syndromic tooth agenesis, divided into hypodontia (n = 24) and oligodontia forms (n = 6), and 42 unaffected family members. Genomic DNA from buccal epithelial cells was used for polymerase chain reaction amplification of functionally important exons of the WNT10A and RUNX2 genes. Direct sequencing reactions were performed to confirm the presence of mutations. The trend of increasing prevalence of WNT10A mutations and a slight increase in the prevalence of RUNX2 mutations were revealed in tooth agenesis cases compared to unaffected family members. There was a higher prevalence of hypodontia than oligodontia, increased frequency of females over males with missing teeth, and a wide phenotypic variability was observed in individuals and families analyzed. The common missense mutations (p.Phe228Ile, p.Arg113Cys, p.Asp217Asn, and p.Gly165Arg) and c.114-56T>C in the WNT10A gene and in-frame-deletion/insertions (11A, 24Q, 30Q), synonymous variant c.240G>A, and 424-33dupC in the RUNX2 gene were identified. These findings highlight an important role of WNT10A and RUNX2 mutations in the genetic etiology of non-syndromic tooth agenesis.

T cells at work: How post-transcriptional mechanisms control T cell homeostasis and activation.

T cells are central players of the adaptive immune system by protecting us from recurring infections and by killing malignant cells. Protective T cell responses rely on the concerted production of effector molecules such as cytolytic mediators, granzymes, and perforins, as well as pro-inflammatory cytokines and chemokines. Once activated, T cells drastically change their gene expression and rapidly respond to insults by producing ample amounts of effector molecules. In the absence of antigen, T cells remain in a quiescent state and survey our body for possible pathogenic insults. Resting T cells are, however, not inert, but continuously regulate their protein production to survive and to be prepared for possible re-infections. Here, we review our current knowledge on the regulation of gene expression in activated and quiescent T cells. We specifically focus on post-transcriptional mechanisms that define the protein output and that allow dormant cells to undergo active signaling and selective translation, keeping them poised for activation. Finally, we discuss which signals drive T cell survival and their preparedness to respond to insults and which mechanisms are involved in these processes.

Detection rates of periodontal bacteria and herpesviruses in different forms of periodontal disease.

The aim was to investigate the detection rates of periodontal bacteria (Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans) and herpesviruses (herpes simplex virus-1 [HSV-1], cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) in different forms and severity of periodontal disease, and to compare them with those in periodontally healthy subjects. One hundred and twenty-nine patients participated in the study: 39 diagnosed with periodontal abscess (PA), 33 with necrotizing ulcerative periodontitis (NUP), 27 with chronic periodontitis (CP), and 30 participants with healthy periodontal tissue represented a healthy control group. All patients with periodontal disease (PA, NUP, and CP) were also divided into two groups according to the severity of their disease: moderate and severe periodontitis. The subgingival samples were collected from the periodontitis active sites and the detection of microorganisms was performed by end-point polymerase chain reaction analyses. The results revealed significantly higher detection rates of P. gingivalis, T. forsythia, and P. intermedia in all three groups of patients with periodontitis than in healthy participants. The highest detection rate of A. actinomycetemcomitans was noticed in CP, which was significantly higher than that in PA, NUP, and healthy control. The occurrence of EBV was significantly higher in NUP than in CP and healthy participants. CMV was detected significantly more frequently in PA and NUP than in CP and healthy participants. Comparisons among healthy participants and patients with moderate and severe periodontitis showed significantly higher detection rates of EBV and CMV in patients with severe forms of periodontitis than in healthy participants and those with moderate periodontitis.

Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production.

Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes.

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b.

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

The complex of MCMV proteins and MHC class I evades NK cell control and drives the evolution of virus-specific activating Ly49 receptors.

CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I-restricted Ly49 receptors.

Clinical antibacterial effectiveness and biocompatibility of gaseous ozone after incomplete caries removal.

OBJECTIVES: To evaluate local effect of gaseous ozone on bacteria in deep carious lesions after incomplete caries removal, using chlorhexidine as control, and to investigate its effect on pulp vascular endothelial growth factor (VEGF), neuronal nitric oxide synthase (nNOS), and superoxide dismutase (SOD). MATERIALS AND METHODS: Antibacterial effect was evaluated in 48 teeth with diagnosed deep carious lesion. After incomplete caries removal, teeth were randomly allocated into two groups regarding the cavity disinfectant used: ozone (open system) or 2% chlorhexidine. Dentin samples were analyzed for the presence of total bacteria and Lactobacillus spp. by real-time quantitative polymerase chain reaction. For evaluation of ozone effect on dental pulp, 38 intact permanent teeth indicated for pulp removal/tooth extraction were included. After cavity preparation, teeth were randomly allocated into two groups: ozone group and control group. VEGF/nNOS level and SOD activity in dental pulp were determined by enzyme-linked immunosorbent assay and spectrophotometric method, respectively. RESULTS: Ozone application decreased number of total bacteria (p = 0.001) and Lactobacillus spp. (p < 0.001), similarly to chlorhexidine. The VEGF (p < 0.001) and nNOS (p = 0.012) levels in dental pulp after ozone application were higher, while SOD activity was lower (p = 0.001) comparing to those in control pulp. CONCLUSIONS: Antibacterial effect of ozone on residual bacteria after incomplete caries removal was similar to that of 2% chlorhexidine. Effect of ozone on pulp VEGF, nNOS, and SOD indicated its biocompatibility. CLINICAL RELEVANCE: Ozone appears as effective and biocompatible cavity disinfectant in treatment of deep carious lesions by incomplete caries removal technique.

Cardiovascular variability and ß-ARs gene expression at two stages of doxorubicin - Induced cardiomyopathy.

Using comprehensive analysis of heart rate (HRV) and blood pressure (BPV) short-term variability we estimated the time course of changes of autonomic nervous system remodeling in two stages of doxorubicin-induced cardiomyopathy (DCM). We also investigated the level of gene expression of cardiac ß-1 (ß-1AR) and ß-2 (ß-2AR) adrenoceptors. Experiments were performed in adult male Wistar rats equipped with indwelling catheters for BP recording and blood withdrawal. A 15 mg/kg total cumulative dose of doxorubicin was injected i.p. to rats to induce DCM or saline for control (n=18). Rats were assessed for general toxicity, cardiovascular hemodynamic and echocardiography before treatment (n=6), 35 days (DOX35; n=6) and 70 days (DOX70; n=6) post-treatment. HRV was evaluated by spectral analysis, Poincaré plots, sample and approximate entropy. Expression of ß-1AR and ß-2AR mRNA was evaluated by RT-qPCR. Doxorubicin-treated rats exhibited poor general condition and lower survival than saline-treated rats. In DOX35 rats, there were no echocardiography signs of decompensation, no increase in serum cardiac troponins, but there was an increase of HRV and decrease of HR complexity. In these rats typical microscopic signs of cardiotoxicity were seen along with over-expression of ß-1AR mRNA. 70 days post-treatment echocardiography revealed signs of decompensation and serum cardiac troponin T was increased. At this stage BPV decreased. In conclusion, HRV increase matches transient over-expression of cardiac ß-1AR mRNA in compensate stage of DCM while decompensate stage of DCM is characterized by a decrease of BPV and no changes in ß-1AR and ß-2AR gene expression.

Association of C35T polymorphism in dihydrofolate reductase gene with toxicity of methotrexate in rheumatoid arthritis patients.

BACKGROUND: Methotrexate (MTX), a folate analogue, is the most commonly used disease-modifying drug in the treatment of rheumatoid arthritis (RA). However, high interindividual differences in drug response are present among RA patients. RESEARCH DESIGN AND METHODS: In a group of 234 RA patients treated with MTX, we investigated whether rs1650697 polymorphism in DHFR gene may have an impact on MTX efficacy and/or adverse drug effects (ADEs). Relative DAS28 values (rDAS28) were used for the estimation of MTX therapy and all ADEs were recorded. Patients were genotyped for selected polymorphism by real-time PCR method. RESULTS: According to the European League Against Rheumatism criteria after 6 months of MTX therapy, 196 patients (83.8%) were classified as responders (25 (10.7%) were good and 171 (73.1%) were moderate) and 38 patients (16.2%) as nonresponders. ADEs were observed in 55 patients (23.5%). CONCLUSIONS: Our results showed that the presence of T allele might be protective against MTX hepatotoxicity measured by transaminase levels (p = 0.05). Furthermore, among patients who also received low-dose corticosteroids, we have found a lower rDAS value in patients with CC genotype (p = 0.039).

The influence of ageing on the extrapineal melatonin synthetic pathway.

Ageing affects various physiological and metabolic processes in a body and a progressive accumulation of oxidative damage stands out as often used explanation. One of the most powerful scavenger of reactive oxygen species (ROS) in all organs is melatonin. A majority of melatonin supplied to the body via blood originates from the pineal gland. However, we have been interested in a locally produced melatonin. We have used 2.5- and 36-months-old Wistar rats. Tissues were collected and gene expression of AA-NAT and ASMT, the two key enzymes in a synthesis of melatonin, was determined in brain, liver, kidney, heart, skin, and intestine. Since melatonin can influence antioxidant enzymes, the activity of superoxide dismutase (SOD) and catalase (CAT), and the level of GSH were measured in liver. In addition, Copper (Cu), Zinc (Zn), and Manganese (Mn) were also determined in liver since these microelements might affect the activity of antioxidant enzymes. The expression of AA-NAT and ASMT was increased in liver and skin of old animals. A positive correlation in AA-NAT and ASMT expression was observed in liver, intestine and kidney. Moreover, the activity of CAT enzyme in liver was increased while SOD activity was decreased. SOD and CAT were probably affected by the observed decreased amount of Cu, Zn, and Mn in liver of old animals. In our model, extrapineal melatonin pathway in ageing consisted of complex interplay of locally produced melatonin, activities of SOD and CAT, and adequate presence of Cu, Zn and Mn microelements in order to defend organs against oxidative damage.

IL-1R8 is a checkpoint in NK cells regulating anti-tumour and anti-viral activity.

Interleukin-1 receptor 8 (IL-1R8, also known as single immunoglobulin IL-1R-related receptor, SIGIRR, or TIR8) is a member of the IL-1 receptor (ILR) family with distinct structural and functional characteristics, acting as a negative regulator of ILR and Toll-like receptor (TLR) downstream signalling pathways and inflammation. Natural killer (NK) cells are innate lymphoid cells which mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses. NK cells mediate resistance against haematopoietic neoplasms but are generally considered to play a minor role in solid tumour carcinogenesis. Here we report that IL-1R8 serves as a checkpoint for NK cell maturation and effector function. Its genetic blockade unleashes NK-cell-mediated resistance to hepatic carcinogenesis, haematogenous liver and lung metastasis, and cytomegalovirus infection.

NCR1-deficiency diminishes the generation of protective murine cytomegalovirus antibodies by limiting follicular helper T-cell maturation.

NKp46/NCR1 is an activating NK-cell receptor implicated in the control of various viral and bacterial infections. Recent findings also suggest that it plays a role in shaping the adaptive immune response to pathogens. Using NCR1-deficient (NCR1gfp/gfp ) mice, we provide evidence for the role of NCR1 in antibody response to mouse cytomegalovirus infection (MCMV). The absence of NCR1 resulted in impaired maturation, function and NK-cell migration to regional lymph nodes. In addition, CD4+ T-cell activation and follicular helper T-cell (Tfh) generation were reduced, leading to inferior germinal center (GC) B-cell maturation. As a consequence, NCR1gfp/gfp mice produced lower amounts of MCMV-specific antibodies upon infection, which correlated with lower number of virus-specific antibody secreting cells in analyzed lymph nodes.

Mouse cytomegalovirus encoded immunoevasins and evolution of Ly49 receptors - Sidekicks or enemies?

Cytomegaloviruses (CMVs) have dedicated a large portion of their genome towards immune evasion targeting many aspects of the host immune system, particularly NK cells. However, the host managed to cope with the infection by developing multiple mechanisms to recognize viral threat and counterattack it, thus illustrating never-ending evolutionary interplay between CMV and its host. In this review, we will focus on several mechanisms of NK cell evasion by mouse CMV (MCMV), the role of host inhibitory and activating Ly49 receptors involved in the virus control and acquisition of adaptive features by NK cells as a consequence of MCMV infection.

IL-33/ST2 pathway drives regulatory T cell dependent suppression of liver damage upon cytomegalovirus infection.

Regulatory T (Treg) cells dampen an exaggerated immune response to viral infections in order to avoid immunopathology. Cytomegaloviruses (CMVs) are herpesviruses usually causing asymptomatic infection in immunocompetent hosts and induce strong cellular immunity which provides protection against CMV disease. It remains unclear how these persistent viruses manage to avoid induction of immunopathology not only during the acute infection but also during life-long persistence and virus reactivation. This may be due to numerous viral immunoevasion strategies used to specifically modulate immune responses but also induction of Treg cells by CMV infection. Here we demonstrate that liver Treg cells are strongly induced in mice infected with murine CMV (MCMV). The depletion of Treg cells results in severe hepatitis and liver damage without alterations in the virus load. Moreover, liver Treg cells show a high expression of ST2, a cellular receptor for tissue alarmin IL-33, which is strongly upregulated in the liver of infected mice. We demonstrated that IL-33 signaling is crucial for Treg cell accumulation after MCMV infection and ST2-deficient mice show a more pronounced liver pathology and higher mortality compared to infected control mice. These results illustrate the importance of IL-33 in the suppressive function of liver Treg cells during CMV infection.

Murine Cytomegalovirus Infection Induces Susceptibility to EAE in Resistant BALB/c Mice.

In contrast to C57BL/6 mice, BALB/c mice are relatively resistant to the induction of experimental autoimmune encephalomyelitis (EAE) after challenge with MOG35-55 peptide. Here, we provide the first evidence that infection with murine cytomegalovirus (MCMV) in adulthood abrogates this resistance. Infected BALB/c mice developed clinical and histological signs similar to those seen in susceptible C57BL/6 mice. In addition to CD4+ cells, large proportion of cells in the infiltrate of diseased BALB/c mice was CD8+, similar with findings in multiple sclerosis. CD8+ cells that responded to ex vivo restimulation with MOG35-55 were not specific for viral epitopes pp89 and m164. MCMV infection favors proinflammatory type of dendritic cells (CD86+CD40+CD11c+) in the peripheral lymph organs, M1 type of microglia in central nervous system, and increases development of Th1/Th17 encephalitogenic cells. This study indicates that MCMV may enhance autoimmune neuropathology and abrogate inherent resistance to EAE in mouse strain by enhancing proinflammatory phenotype of antigen-presenting cells, Th1/Th17, and CD8 response to MOG35-55.

A Viral Immunoevasin Controls Innate Immunity by Targeting the Prototypical Natural Killer Cell Receptor Family.

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.

The role of TERT-CLPTM1L SNPs, hTERT expression and telomere length in the pathogenesis of oral squamous cell carcinoma.

The aim of this study was to assess TERT-CLPTM1L single-nucleotide polymorphisms (SNPs) (rs402710 C/T in the CLPTM1L gene; rs2736100 A/C and rs2736098 G/A in the TERT gene) as risk factors for development of oral squamous cell carcinoma (OSCC), and to investigate the relationship between the analyzed polymorphisms, relative telomere length (RTL), telomerase expression and clinicopathologic characteristics of OSCC in a Serbian population. Paraffin-embedded tumor samples and buccal swabs from cancer-free controls were genotyped using PCR-RFLP, while tumor RTL values and telomerase expression were estimated by real-time PCR and immunohistochemistry, respectively. CLPTM1L rs402710 and TERT rs2736100 polymorphisms were associated with a significantly increased risk of OSCC, and TERT rs2736098 with a significantly decreased risk. No significant association was found between TERT-CLPTM1L polymorphisms, tumor RTL values, telomerase expression, and clinicopathologic features, although a trend towards longer telomeres was evident in telomerase-positive samples and less advanced tumors. Kaplan-Meier survival analysis showed that patients with longer telomeres in their tumors had significantly better overall survival than patients with shorter telomeres. Our research seems to provide strong evidence for an association between CLPTM1L rs402710C/T and TERT rs2736100A/C SNPs and the risk of OSSC, and suggests that higher tumor RTL values and positive hTERT expression may be applicable as early prognostic markers.(J Oral Sci 58, 449-458, 2016).

Estimation of total bacteria by real-time PCR in patients with periodontal disease.

INTRODUCTION: Periodontal diseases are associated with the presence of elevated levels of bacteria within the gingival crevice. OBJECTIVE: The aim of this study was to evaluate a total amount of bacteria in subgingival plaque samples in patients with a periodontal disease. METHODS: A quantitative evaluation of total bacteria amount using quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 20 samples of patients with ulceronecrotic periodontitis and on 10 samples of healthy subjects. The estimation of total bacterial amount was based on gene copy number for 16S rRNA that was determined by comparing to Ct values/gene copy number of the standard curve. RESULTS: A statistically significant difference between average gene copy number of total bacteria in periodontal patients (2.55 x 107) and healthy control (2.37 x 106) was found (p = 0.01). Also, a trend of higher numbers of the gene copy in deeper periodontal lesions (> 7 mm) was confirmed by a positive value of coefficient of correlation (r = 0.073). CONCLUSION: The quantitative estimation of total bacteria based on gene copy number could be an important additional tool in diagnosing periodontitis.

Intrinsic Contribution of Perforin to NK-Cell Homeostasis during Mouse Cytomegalovirus Infection.

In addition to their role as effector cells in virus control, natural killer (NK) cells have an immunoregulatory function in shaping the antiviral T-cell response. This function is further pronounced in perforin-deficient mice that show the enhanced NK-cell proliferation and cytokine secretion upon mouse cytomegalovirus (MCMV) infection. Here, we confirmed that stronger activation and maturation of NK cells in perforin-deficient mice correlates with higher MCMV load. To further characterize the immunoregulatory potential of perforin, we compared the response of NK cells that express or do not express perforin using bone-marrow chimeras. Our results demonstrated that the enhanced proliferation and maturation of NK cells in MCMV-infected bone-marrow chimeras is an intrinsic property of perforin-deficient NK cells. Thus, in addition to confirming that NK-cell proliferation is virus load dependent, our data extend this notion demonstrating that perforin plays an intrinsic role as a feedback mechanism in the regulation of NK-cell proliferation during viral infections.