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MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.

Alleles

Restriction insensitivity in bacteriophage T5 I. Genetic characterization of mutants sensitive to EcoRI restriction.

Unmodified bacteriophage T5 is able to grow normally on bacterial hosts carrying three different Escherichia coli restriction systems, EcoK, EcoPI, and EcoRI. Under the same conditions, the plating efficiency of bacteriophage gamma is less than 10(-9). At least in the case of EcoRI, this lack of in vivo restriction is not due to lack of restriction sites on the T5 DNA molecule. These observations suggest that bacteriophage T5 specifies one or more restriction protection systems. Mutants (ris) of T5 have been isolated which confer sensitivity to EcoRI restriction but not to EcoK or EcoPI. The mutations are located in the pre-early region of the genetic map but are too far apart to be alleles of a single gene. Complementation studies show that the ris mutants can be helped to grow on the EcoRI-restricting host by coinfection with T5+. This result provides evidence for a restriction protection function but does not necessarily show that the ris mutants are defective in such a system.

Coliphages

Early-life food restriction of broiler chickens. I. Methods of application, amino acid supplementation and the age at which restrictions should commence.

1. Three experiments were performed to examine practical methods of early-life food restrictions and to examine the influence of increased dietary lysine or methionine on bodyweight recovery during re-alimentation in broiler chickens. 2. Food intake of broiler chicks was restricted quantitatively or by dietary dilution at 3 ages and the birds slaughtered at 49 d of age. The success of each food restriction was measured in terms of bodyweight recovery and body fat content. 3. Discontinuous food restriction and restriction by dietary dilution produced similar effects to a previously established food restriction regime. Bodyweight recovery was complete and fat contents depressed. FCR was inconsistently affected. 4. Supplementation of the finisher diet with lysine and/or methionine produced no conclusive results when this diet was fed to broilers previously subjected to early-life food restriction.

Adipose Tissue

Effect of dietary protein restriction or food restriction on oxygen consumption and mitochondrial distribution in cardiac and red and white skeletal muscle of rats.

The effects of food restriction and dietary protein restriction on the oxygen consumption of three types of muscle in the rat were compared. Control rats were fed a diet containing 27% casein. Two groups of protein-restricted rats were fed isoenergy diets containing 15% or 8% casein. Two groups of food-restricted rats were fed either the 27% or 15% casein diets in amounts equivalent to 35% of the intake of energy. Oxygen consumption was measured in papillary muscle and in fibers from the red (high oxidative) and white (low oxidative) portions of the quadriceps with pyruvate and malate as substrates. Control values (micronlO2/g dry wt/min) were 35.2 + 1.5 (papillary), 20.7+/-1.9 (red), and 7.8+/-0.7 (white). Restriction of dietary protein content failed to alter the oxygen consumption of any of the muscle types. In contrast, involuntary food restriction,combined with a reduction in protein content, caused a reduction in the oxygen consumption of papilary and white skeletal muscle, but the metabolism of red skeletal muscle was not affected. No differences were found in the appearance and distribution of mitochondria in cardiac or red skeletal muscle after examination by electron microscopy, but a depletion of subsarcolemmal mitochondria was evident in white muscle from the food-restricted rats.

Animals

Characterization of Neisseria meningitidis isolated by ribosomal RNA gene restriction patterns and restriction endonuclease digestion of chromosomal DNA.

The use of ribosomal RNA (rRNA) gene restriction patterns to study the molecular epidemiology of Neisseria meningitidis was investigated. Ninety-four isolates of Neisseria meningitidis were characterized by their rRNA gene restriction patterns with 16 + 23 S rRNA from Escherichia coli as a probe. Thirteen rRNA gene restriction patterns were recognized; each of these patterns represented between 1 and 30 isolates. Isolated with the outbreak-associated phenotype B15P1.16 (sulphonamide resistant) all gave a single rRNA gene restriction pattern but this pattern also contained isolates with other phenotypes. Further discrimination between isolates was achieved by comparison of banding patterns resulting from restriction endonuclease digestion of chromosomal DNA with Bgl II. This gave a banding pattern consisting of about ten bands which was simple to interpret. Using this technique 94 isolates were classified in 54 patterns containing between 1 and 14 isolates. Restriction endonuclease analysis with Bgl II characterized outbreak-associated isolates with the phenotype B15P1.16 and enabled strains not typable by conventional methods to be identified as probable outbreak-associated isolates. The techniques should prove useful for epidemiological studies.

Bacterial Proteins

Early-life food restriction of broiler chickens. II. Effects of food restrictions on the development of fat tissue.

1. The physiological and metabolic effects of a short term food restriction of broiler chickens at an early age and grown to 49 d of age were examined. 2. Fat accretion was measured by tritium dilution. Adipocyte characteristics of the fat tissue were examined by cell counting after fixation with osmium tetroxide and the metabolic effects of food restrictions were measured by closed-circuit respiration calorimetry. 3. The success of food restrictions in allowing full bodyweight recovery and producing decreases in body fat was associated with a negative energy balance and a positive nitrogen balance achieved during the restriction phase. 4. Food restrictions produced decreases in body fat possibly by causing a delay in adipocyte hyperplasia. Excess dietary energy was then lost as heat until adipocyte hyperplasia re-commenced allowing the bird to store surplus dietary energy as fat. 5. A food restriction that allows full bodyweight recovery and maximum carcase fat reduction in the broiler chicken at 49 d may not be appropriate for birds grown for longer periods.

Adipose Tissue

Dietary fat level as affecting running performance and other performance-related parameters of rats restricted or non-restricted in food intake.

The effect of food energy density on certain physical performance characteristics of rats was studied during ad libitum and weight-restricted feeding. After a 16-week treadmill training period, 48 young adult male Long-Evans rats were divided into six groups, receiving 20%, 40% or 70% of energy as dietary fat and fed ad libitum or 40% by weight of average ad libitum intake. They were run to exhaustion once a week. Increased dietary fat level was not correlated with superior running performance at either food intake level, although during restricted feeding the high fat group received 40% more energy than the low fat group. Lower body weight was correlated with increased running performance at both food intake levels. The rate of performance increase with decreasing body weight during food restriction was significantly higher for the low fat than for the high fat group. Water consumption was considerably lower during restricted than during ad libitum feeding. Plasma glucose was lower when the 70% fat diet was fed than when compared to the other fat diets at both levels of food intake, lower from day 8 on of restricted that during ad libitum food intake, and lower on day 15 than on day 8 of the restricted intake.

Animals

Restricted feeding and broiler performance: age at initiation and length of restriction.

Broiler cockerels were provided either ad libitum access or restricted to alternate-day access to feed initiated at different ages and lasting for different durations. Regimens consisted of ad libitum feeding; alternate-day feeding; alternate-day feeding from 0 to 6, 6 to 12, or 12 to 18 days of age (DOA); alternate-day feeding from 0 to 12, 0 to 6, and 12 to 18, or 6 to 18 DOA; and alternate-day feeding from 0 to 18 DOA. By 39 DOA, chicks restricted for only one 6-day period reached body weights equivalent to those of chicks eating ad libitum. Body weights of chicks restricted for more than 6 days during the first 18 days after hatch were lower than those of chicks eating ad libitum at 39 DOA. Restriction for more than 6 days improved feed efficiency. Chicks restricted for the whole experiment had superior feed efficiency but low body weights. Antibody responses to sheep erythrocyte antigen were similar among feeding regimens. Response to Escherichia coli inoculation was generally less severe in chicks provided restricted access to feed, and overall mortality was highest in chicks eating ad libitum. Feeding regimens had little effect on organ weights relative to body weight, amount of abdominal fat (either on an absolute basis or relative to body weight), or percentage lipid in the abdominal fat pad.

Abdomen

A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA.

Genetic analyses of DNA restriction and modification mechanisms have been encumbered by the inability to rigorously select for mutant phenotypes associated with these systems. The application of restriction endonucleases has now proved to be a successful approach to the genetic analyses of small genomes that are recalcitrant to the more standard genetic techniques. Restriction endonucleases EcoRI and HindIII were used to analyze the structure of the plasmid genome responsible for the EcoRI restriction endonuclease and modification methylase. This plasmid in the original clinical isolate of Escherichia coli appears to be identical to the ColE 1 plasmid except for a 1.95 kilobase pair segment which contains these genes. A preliminary restriction map of this plasmid is presented.

Base Sequence

Bvr-1, a restriction locus of a type C RNA virus in the feline cellular genome: pleiotropic restriction of endogenous BALB virus in cat X mouse somatic cell hybrids.

Bvr-1 is a dominant X-linked feline gene which restricts the replication of B-tropic murineleukemia virus (B-MuLV) in somatic cell hybrids between murine BALB/c-RAG cells and FL-74 feline cells. Since the hybrids were originally derived by the hypoxanthine aminopterin thymidine selection scheme, counter selection experiments on 6-thioguanine result in preferential survival of hybrid cells which have spontaneously lost the feline X-chromosome on which is located the structural gene for hypoxanthine guanine phosphoribosyl transferase (IMP: pyrophosphate phosphoribosyl transferase, E.C. 2.4.2.8) and Bvr-1. Back selected Bvr-1- cells express high parental levels of B-MuLV. Bvr-1 effectively restricts the IdU-mediated induction of the endogenous xenotropic BALB virus (BALB: virus 2) but not the endogenous N-tropic virus (BALB: virus 1). Pleiotropic restriction of B-MuLV and X-MuLV, but not N-MuLV suggests that the viral targets of Bvr-1 (either viral components or functions in viral assembly) of the B-tropic and X-tropic endogenous BALB viruses are similar to each other but distinct from the target in the N-tropic virus. Very low levels of B-MuLV are detected in restricted cells, but this residual virus is not infectious in either NIH-3T3 or BALB-3T3 mouse cells which are genotypically Fv-1N/Fv-1N and Fv-1B/Fv-1B, respectively. Passage of residual virus through host cells without Fv-1 related restriction (SC-1) results in production of infectious B-MuLV indistinguishable from that produced by RAG parent cells.

Animals

Restricted helper function of F1 hybrid T cells positively selected to heterologous erythrocytes in irradiated parental strain mice. II. Evidence for restrictions affecting helper cell induction and T-B collaboration, both mapping to the K-end of the H-2 complex.

Studies with H-2-congenic and recombinant strains showed that when F1 hybrid T cells were activated to sheep erythrocytes in irradiated mice of parental strain or related strain, a population of helper cells was generated which collaborated only with B cells sharing the K-end of the H-2 complex with the strain used for activation. No evidence was found that the restriction in helper function (a) reflected a deficiency of appropriate macrophages during T-B collaboration, or (b) was influenced by the Ig allotype of the B cells. It was concluded that the results signified restrictions acting at both the level of helper cell induction (presumed to be a reflection of T-macrophage interactions in the irradiated intermediate hosts) and during T-B collaboration. With (CBA X C57BL/6)F1 cells, the restrictions at each level mapped to the same region i.e. to the left of the I-B subregion. Consequently, one gene (or set of genes) might control restriction at both levels. If so, T-cell recognition of major histocompatibility complex-associated antigen on macrophages and on specific B cells would be either identical or very similar. The fact that genes mapping to the K-end of the H-2 complex also control the restrictive interactions of homozygous T cells implies that F1 T cells behave functionally as a mixture of T cells derived from the two parental strains. Positive selection to antigen in parental strain mice appears simply to alter the ratio of these two populations.

Animals

MHC restriction of synovial fluid lymphocyte responses to the triggering organism in reactive arthritis. Absence of a class I-restricted response.

Synovial fluid mononuclear cells (SFMC) from patients with reactive arthritis (ReA) show marked proliferative responses to preparations of the organism triggering the arthritis. Initial studies with MHC-specific MoAbs have indicated that a significant element of these proliferative responses is mediated by class II MHC-restricted CD4+ T cells. It is imperative to establish the presence or absence of a class I-restricted response, for two reasons. Firstly, the association of ReA with the MHC class I molecule, HLA B27, raises the possibility of there being a B27-restricted response to the triggering organism. Secondly, a number of the organisms associated with ReA are intracellular pathogens, whose antigens might be expected to be presented by class I MHC molecules. In an effort to identify a class I MHC-restricted pathogen-specific response in the SFMC of ReA patients, we have assessed the proliferative responses of SFMC depleted of CD4+ T cells. Responses were grossly diminished by CD4+ T cell depletion. We also investigated Chlamydia-specific cytotoxicity in the SFMC of patients with sexually acquired ReA in a system using productive chlamydial infection to produce both targets and effectors. Significant antigen specific cytotoxicity was not seen. These experiments do not provide evidence to support the existence of pathogen-specific responses by CD8+, class I-restricted synovial fluid T cells in ReA.

Arthritis, Reactive

Host-dependent restriction of mengovirus replication. II. Effect of host restriction on late viral RNA synthesis and viral maturation.

Restricted mengovirus replication in Mandin-Darby bovine kidney (MDBK) cells is characterized by a 400-fold reduction in infectious virus yield and a 40-fold increase in the production of noninfectious virus. Using conditions which insure that all MDBK cells are infected, virus-specific RNA and protein synthesis were measured in the restrictive host and in a permissive host for mengovirus, HeLa cells. Labeling kinetics and sucrose gradient analysis of mengovirus-specific RNA from MDBK cells show a reduction of 10-fold in virion RNA, 5-fold in double-stranded RNA, and 12.5-fold in single-stranded RNA. The viral RNA biosynthetic processes which occur late in the replicative cycle and result in the production of 90% of the single-stranded viral RNA that is packaged into capsid proteins in the permissive host are absent in restrictive MDBK cells. Viral protein synthesis as measured by labeled viral-specific polysome is decreased, and there is an accumulation of 80S subviral particles in the restricted host. It is suggested that restriction may act at a number of stages of viral replication and maturation.

Amino Acids

Body composition, nitrogen metabolism, and energy utilization with feeding of mildly restricted (4.2 MJ/d) and severely restricted (2.1 MJ/d) isonitrogenous diets.

To determine the effects on weight loss of feeding isonitrogenous diets in mildly restricted (4.2 MJ/d) and severely restricted (2.1 MJ/d) amounts, we measured body composition, weight loss-energy deficit ratio, and nitrogen metabolism in 14 obese women housed in a metabolic ward consuming hypoenergetic diets for 21 d. Subjects consumed either a 4.2-MJ/d diet (50 g protein, 175 g carbohydrate) or a 2.1-MJ/d diet (50 g protein, 75 g carbohydrate). Body composition and leucine oxidation and turnover were determined before and after weight loss. Energy deficit was calculated from resting metabolic rates. Subjects fed the 2.1-MJ/d diet showed a greater weight loss (6.1 +/- 0.5 vs 4.5 +/- 0.5 kg; mean +/- SE, P less than 0.05) and fat loss (3.9 +/- 0.3 vs 3.0 +/- 0.3 kg, P less than 0.05). Weight loss-energy deficit ratio was the same with both diets. Nitrogen balance and leucine oxidation and turnover were similar in both groups. We conclude that with feeding of isonitrogenous hypoenergetic diets, severe restriction of energy content (2.1 MJ/d, 75 g carbohydrate) will enhance weight and fat loss without increasing nitrogen loss compared with mild restriction of energy (4.2 MJ/d).

Body Composition

Overlapping epitopes that are recognized by CD8+ HLA class I-restricted and CD4+ class II-restricted cytotoxic T lymphocytes are contained within an influenza nucleoprotein peptide.

Viral epitopes that are recognized by both HLA class I-restricted and class II-restricted T cells have been defined for a type A influenza virus nucleoprotein (NP) peptide. CD8+ and CD4+ CTL lines have been generated against a synthetic peptide encompassing residues 335 to 349 of NP that are restricted by HLA-B37 and HLA-DQw5, respectively. Both of these CTL populations were capable of specifically lysing influenza A virus-infected targets, indicating that a naturally processed NP peptide(s) was being mimicked by the NP (335-349) peptide. Amino acid residues that are critical for recognition of this NP determinant in the context of HLA-B37 and HLA-DQw5 were investigated by the use of panels of truncated and alanine-substituted NP peptides. The results demonstrate that: 1) truncations in the amino- or carboxy-terminal ends differentially affect CD8+ and CD4+ CTL recognition; 2) the NP (335-349) sequence contains two octapeptide epitopes that share a core of six amino acid residues (NP 338-343); and 3) alanine substitutions at five of these residues abrogated recognition by at least one of the CD8+ and CD4+ CTL lines. Thus, these class I- and class II-restricted CTL lines recognize similar but distinct epitopes, and different structural features of the NP peptide are required for presentation by HLA-B37 and HLA-DQw5. Comparison of the amino acid sequences of the NP peptide presented by HLA-B37 and HLA-DQw5 with other peptides known to be presented by both class I and class II molecules revealed a common motif among these peptides.

Amino Acid Sequence