Cmv1 and natural killer cell responses to murine cytomegalovirus infection.

The dissection of genetic resistance to murine cytomegalovirus infection in inbred laboratory mouse strains led to the identification of a natural killer cell activation receptor that recognizes a virus-encoded protein. Herein, we summarize the genetic approach and findings that have provided novel insights into innate immune control of virus infections.

Natural killer gene complex (Nkc) allelic variability in inbred mice: evidence for Nkc haplotypes.

Allelic variability for mouse Chromosome 6 Nkc loci was assessed in 22 common laboratory strains of mice using selected natural killer gene complex (Nkc)-linked sequence tagged site markers. Most Nkc markers distinguished three or more alleles for a particular locus in the assessed mouse strains. Nkc locus alleles were highly conserved among genealogically related inbred strains, whereas far less similarity was observed among unrelated strains. Concurrent strain-to-strain comparisons for all Nkc-linked loci revealed common and uncommon Nkc haplotypes, including some that were likely recombinant. Nkc allele and haplotype assignments in inbred mouse strains and correlation with phenotypic traits should facilitate positional gene cloning strategies for unknown Nkc-linked trait modification loci.

CD1d-restricted NKT cells: an interstrain comparison.

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.

Vital involvement of a natural killer cell activation receptor in resistance to viral infection.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Cutting edge: a NK complex-linked locus governs acute versus latent herpes simplex virus infection of neurons.

Herpes simplex causes latent infections that periodically reactivate. Specific immunization attempts are failing to control herpes, prompting a fresh look at which host responses predominate. We report a NK complex-linked genetic locus, Rhs1, whose alleles influence the magnitude of experimental herpes simplex. Rhs1 provided rapid control of primary infection but caused a reciprocal increase in the number of latently infected neurons. Thus, in principle, establishment of latency is a consequence of efficient front line defense against herpesvirus infection. Based on conservation between human and mouse NK complexes, the data predict the presence of a human Rhs1 orthologue on chromosome 12p12-13.

NK1.1+ cells and murine cytomegalovirus infection: what happens in situ?

NK cells mediate early host defense against viral infection. In murine CMV (MCMV) infection NK cells play a critical role in controlling viral replication in target organs, such as spleen and liver. Until now it has not been possible to directly examine the role of NK cells in MCMV-induced inflammation in situ due to the inability to stain specifically for NK cells in infected tissues. In this study, we describe a method of in vivo fixation, resulting in the first identification of NK cells in situ using NK1.1 as the marker. Using this method, we characterize the NK1.1(+) cellular component of the inflammatory response to wild-type MCMV in the spleen, liver, and lung of genetically susceptible and resistant mice following i.p. infection. This study provides the first in situ description of the cellular response mediated specifically by NK cells following MCMV infection.

Why do cultured transplanted myoblasts die in vivo? DNA quantification shows enhanced survival of donor male myoblasts in host mice depleted of CD4+ and CD8+ cells or Nk1.1+ cells.

Overcoming the massive and rapid death of injected donor myoblasts is the primary hurdle for successful myoblast transfer therapy (MTT), designed as a treatment for the lethal childhood myopathy Duchenne muscular dystrophy. The injection of male myoblasts into female host mice and quantification of surviving male DNA using the Y-chromosome-specific (Y1) probe allows the speed and extent of death of donor myoblasts to be determined. Cultured normal C57BL/10Sn male donor myoblasts were injected into untreated normal C57BL/10Sn and dystrophic mdx female host mice and analyzed by slot blots using a 32P-labeled Y1 probe. The amount of male DNA from donor myoblasts showed a remarkable decrease within minutes and by 1 h represented only about 10-18% of the 2.5 x 10(5) cells originally injected (designated 100%). This declined further over 1 week to approximately 1-4%. The host environment (normal or dystrophic) as well as the extent of passaging in tissue culture (early "P3" or late "P15-20" passage) made no difference to this result. Modulation of the host response by CD4+/CD8+ -depleting antibodies administered prior to injection of the cultured myoblasts dramatically enhanced donor myoblast survival in dystrophic mdx hosts (15-fold relative to untreated hosts after 1 week). NK1.1 depletion also dramatically enhanced donor myoblast survival in dystrophic mdx hosts (21-fold after 1 week) compared to untreated hosts. These results provide a strategic approach to enhance donor myoblast survival in clinical trials of MTT.

Poison prevention counseling: a comparison between family practitioners and pediatricians.

OBJECTIVES: To compare the current opinions and practices of family practitioners with those of pediatricians regarding poison prevention anticipatory guidance (PPAG) and to further assess which factors, if any, are associated with providing counseling on this topic. DESIGN: Cross-sectional mail survey. PARTICIPANTS: Family practitioners and pediatricians throughout the United States and Puerto Rico, randomly selected from the membership list of the American Medical Association. MAIN OUTCOME MEASURE: Reported frequency of providing parents with PPAG. RESULTS: Of the 1000 physicians surveyed, 975 were eligible and 500 returned surveys that could be analyzed (227 family practitioners and 273 pediatricians), for a response rate of 51.3%. The majority of physicians in each field (family practice, 81.9%; pediatrics, 87.2%) reported that they believe it is their responsibility to provide PPAG to parents (P = .08). Family practitioners, however, were less likely than pediatricians to provide parents with PPAG (66.5% vs 91.9%; P<.001). When adjusted for other variables, such as age and sex, family practitioners were 5.4 times less likely than pediatricians to provide parents with PPAG (odds ratio, 0.19; 95% confidence interval, 0.09-0.37). Family practitioners, more often than pediatricians, cited lack of training on poisoning prevention as a reason for not providing parents with PPAG (46.1% vs 18.2%; P = .02). Among all physicians, those who received postresidency training on PPAG were more likely to provide PPAG than those who had not received postresidency training on this topic (odds ratio, 3.21; 95% confidence interval, 1.44-7.18). Having received residency training on poisoning prevention, however, did not increase the likelihood of providing PPAG (odds ratio, 1.69; 95% confidence interval, 0.86-3.30). CONCLUSIONS: Although it is currently recommended to include PPAG as part of the routine preventive pediatric care, this study shows that one third of family practitioners do not provide parents with PPAG. Family practitioners should increase their efforts aimed at poisoning prevention. Those involved with training residents in family practice and pediatrics should place greater emphasis on this topic to increase the impact of this training on actual PPAG practices.

Localization on a physical map of the NKC-linked Cmv1 locus between Ly49b and the Prp gene cluster on mouse chromosome 6.

The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of approximately 390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.

Perforin is a major contributor to NK cell control of tumor metastasis.

We provide the first demonstration, using experimental and spontaneous models of metastasis in C57BL/6 (B6) (RM-1 prostate carcinoma) and BALB/c (DA3 mammary carcinoma) mice, that tumor metastasis is primarily controlled by perforin-dependent cytotoxicity mediated by NK1.1+ cells. MHC class Ilow RM-1 and DA3 tumor cells were sensitive in vitro to Fas-mediated lysis or spleen NK cells in a perforin-dependent fashion. Perforin-deficient NK cells did not lyse these tumors, and perforin-deficient mice were 10-100-fold less proficient than wild-type mice in rejecting the metastasis of tumor cells to the lung. Fas ligand mutant gld mice displayed uncompromised protection against tumor metastasis. Depletion of NK subsets resulted in greater numbers of metastases than observed in perforin-deficient mice, suggesting that perforin-independent effector functions of NK cells may also contribute to protection from tumor metastasis.

The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing.

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6-Cmv1(r) congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4(+) B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.

Peptide based cytotoxic T-cell vaccines; delivery of multiple epitopes, help, memory and problems.

Synthetic CD8+ cytotoxic T-lymphocyte (CTL) peptide epitope based vaccines are being developed against a number of human diseases. Here we describe extensive preclinical testing of peptide epitope vaccines formulated with a protein as a source of CD4 help and Montanide ISA 720, an adjuvant currently in human clinical trials. Such water-in-oil formulations could effectively co-deliver several peptide epitopes and simultaneously induce multiple independent CTL responses. The efficiency of CTL induction by some peptides was, however, dependent on the aqueous buffer conditions, with poor performance correlating with non-covalent peptide oligomerisation. Any of a number of proteins currently used in human vaccines could supply CD4 help and no difference in CTL induction was obtained if the CD4 response was amnestic or a primary. Peptide immunisation was found to induce long term CTL memory and the recall of protective responses did not depend on an amnestic CD4 response. Slow pyroglutamic acid formation and rapid oxidation of methionine residues was observed in water-in-oil formulations, however, the latter had no effect on CTL induction. These data highlight the need to monitor for potential deleterious chemical events and interpeptide interactions, but illustrate that peptide based vaccination can effectively deliver multiple epitopes, in conjunction with any protein, and induce protective memory.

Carbon monoxide mass exposure in a pediatric population.

OBJECTIVES: To describe the outcomes of a mass carbon monoxide (CO) intoxication, and to calculate the CO half-life in a pediatric school-aged population. METHODS: A retrospective chart review was performed based on Regional Poison Center database information, hospital laboratory data, and medical records of the pediatric patients who sought care at one of 3 St. Louis area hospitals, after exposure to high levels of CO. Exposures occurred on January 5, 1996, after evidence of a CO leak was discovered at an area elementary school. Charts were reviewed for major demographics, symptoms reported, carboxyhemoglobin (COHb) levels and times, and level of effect. RESULTS: Information about 177 (35%) of the 504 children in attendance at school that day was available. Mean age was 8.7 +/- 1.8 years (range 4-12 years). Symptoms were present in 155 (88%) of the 177 children for whom data were available. Initial COHb levels were obtained for 147 (83.1%) of the 177 children. First mean COHb level was 7.0% (95% CI = 6.6-7.5%). Second COHb level was obtained for 26 children with a mean of 2.7% (95% CI = 2.2-3.2%). Calculated half-life of COHb, on 100% O2 at 1 atm, was 44.0 minutes (95% CI = 39.6-48.2 minutes). CONCLUSION: Some children had symptoms at COHb levels that traditionally have been considered nontoxic. The elimination of COHb was found to be more rapid in this population of children than reported in other studies.

Genetic control of natural killing and in vivo tumor elimination by the Chok locus.

The molecular mechanisms underlying target recognition during natural killing are not well understood. One approach to dissect the complexities of natural killer (NK) cell recognition is through exploitation of genetic differences among inbred mouse strains. In this study, we determined that interleukin 2-activated BALB/c-derived NK cells could not lyse Chinese hamster ovary (CHO) cells as efficiently as C57BL/6-derived NK cells, despite equivalent capacity to kill other targets. This strain-determined difference was also exhibited by freshly isolated NK cells, and was determined to be independent of host major histocompatibility haplotype. Furthermore, CHO killing did not correlate with expression of NK1.1 or 2B4 activation molecules. Genetic mapping studies revealed linkage between the locus influencing CHO killing, termed Chok, and loci encoded within the NK gene complex (NKC), suggesting that Chok encodes an NK cell receptor specific for CHO cells. In vivo assays recapitulated the in vitro data, and both studies determined that Chok regulates an NK perforin-dependent cytotoxic process. These results may have implications for the role of NK cells in xenograft rejection. Our genetic analysis suggests Chok is a single locus that affects NK cell-mediated cytotoxicity similar to other NKC loci that also regulate the complex activity of NK cells.

Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells.

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A- subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.