We have analyzed the nature/content of methylated bases in the nuclear DNA of three unicellular eucaryotes. The pattern of methylation was different for each of the three organisms studied: Saccharomyces cerevisiae contained only 5methylcytosine; Tetrahymena pyriformis contained only N6-methyladenine; and Chlamydomonas reinhardi contained both modified bases.

Understanding the events involved in the activation of human bone marrowderived (B) lymphocytes to antibody formation and secretion has been hampered by the lack of a simple reprDducible method of culture and assay. In the mouse model, induction of primary in vitro antigenic stimulation (1), as well as polyclonal B-cell activation (2, 3), with measurement of single cell antibody production by plaque-forming cell (PFC) ~ assays has allowed for in depth probing of the mechanisms of B-cell activation. Such a simple and reproducible methodology has not been available for human studies. A few reports have appeared describing primary in vitro activation of human B lymphocytes from various organs, particularly tonsil, by soluble or particulate antigens with subsequent measurement of antibody response by a PFC assay (4-8). However, these studies have been quite difficult to reproduce, and subsequent follow-up studies have not appeared. In addition, recent studies have pointed out a potential area of significant artifact in primary in vitro stimulation of human cultures with sheep red blood cells (SRBC) (9). Attempts to establish PFC assays after polyclonal activation of human B lymphocytes have met with similar difficulties (10). No reproducible studies have appeared which demonstrate single cell antibody production by human lymphocytes after activation with polyclonal B-cell activators as measured by a PFC assay.The present report describes the conditions necessary for the in vitro induction of polyclonal activation as well as antigenic stimulation of human tonsillar lymphocytes with measurement of antibody production by a direct PFC assay. The methods are simple and the results reproducible. Critical factors for induction and assay are delineated and areas of potential artifact are described. This system can be readily employed to explore the complex events associated with human B-lymphocyte activation. Materials and MethodsTonsils. Tonsils were obtained from subjects undergoing routine tonsillectomy for chronic tonsillitis. Tissue was placed immediately in ice-cold RPMI-1640 (Grand Island Biological Co., 1 Abbreviations used in this paper: FCS, fetal calf serum; FITC, fluoresceinated isothiocyanate; HBSS, Hanks' balanced salt solution; KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharide; NIP, 4-hydroxy-3-iodo-5-nitrophenylacetic acid; PFC, plaque-forming cell; PPD, purified protein derivative of tuberculin; PWM, pokeweed mitogen; SIII, pneumococcal polysaccharide; TNP, trinitrophenyl.

A culture and assay system for the stimulation of human peripheral blood lymphocytes with polyclonal activators of bone-marrow-derived lymphocytes (B cells), such as pokeweed mitogen and Escherichia coil lipopolysaccharide, and subsequent measurement of single cell antiboy production by a hemolysis-in-gel direct plaque-forming cell assay against sheep erythrocytes has been established. The critical culture requirements have been delineated and a new highly sensitive ultrathin gel assay method has been described. Under these conditions a substantial and highly reproducible plaque-forming cell response was detected in normal human peripheral blood. This system can be readily used to explore the complex events associated with activation of human B cells.Knowledge of the complex mechanisms and events associated with activation of bone-marrow-derived (B) lymphocytes is crucial for our understanding of the various disorders of overor underproduction of antibody in man. In the mouse model, sophisticated probing of the precise mechanisms of B cell activation has been made possible by the availability of simple and reproducible plaque-forming cell (PFC) assays for the measurement of single cell antibody production after primary antigenic stimulation in vitro (1) as well as stimulation by polyclonal B cell activators (2, 3). This type of methodology has generally been lacking in human studies in which immunoglobulin production by lymphocytes in culture is usually measured by radioimmunoassay of culture supernates (4) In order for such a simple and convenient culture and assay system to become established as a feasible approach to the study of human B lymphocyte activation, it must be adaptable to the most easily accessible lymphoid organ, peripheral blood. The present study describes the precise conditions necessary for the successful and reproducible culture and PFC assay of lymphocytes from human peripheral blood stimulated with polyclonal B cell activators. MATERIALS AND* METHODSCell Suspensions. Heparinized venous blood (60-100 ml) was drawn from 15 normal adults, and purified mononuclear cell suspensions (lymphocytes and monocytes) were obtained by standard Hypaque-Ficoll density centrifugation (13).Culture Conditions. Cells were suspended for culture in RPMI-1640 medium ssupplemented with 1% trypticase soy broth, 0.02 M L-glutamine, penicillin, 100 units/ml, streptomycin sulfate, 100,gg/ml, and 10% pooled human AB serum.The human AB serum was absorbed in-the cold multiple times before use in order to remove the antibody against sheep erythrocytes present in variable concentrations in most lots of human serum. Varying densities of cells ranging from 1.5 to 10 X 106 lymphocytes in 2 ml of medium were cultured either in multi-well, flat-bottom plastic plates (1.5 cm diameter wells) or in 17 X 100 mm round-bottom, standup plastic tubes. Cultures were stimulated with varying concentrations of either pokeweed mitogen or Eschertchia coli 0128:B12 lipopolysaccharide. Also, separate cultures were stimulated with var...

As part of our studies of the class II genes of miniature swine, we have isolated and characterized cDNA clones corresponding to DRB genes from two major histocompatibility complex homozygous strains. Comparison of the sequences of these clones to those of human DRB genes revealed a striking amino acid homology between the hypervariable residues of SLA-DRBC and the human DRBI-0101 allele. The percentage of differences in these residues between the pig DRBC allele and the human DRBI-OlO allele was significantly lower (29%) than that between the DRBI-OO0 allele and all other human alleles (average, 66.2%). This similarity was not seen in a comparison of the number of silent substitutions, by which the swine DRBC and the human DRB-0101 differed. Since phenotypic selection operates at the level of protein products rather than nucleotide sequences, these data suggest the existence of selective mechanisms that have resulted in similar hypervariable regions in certain alleles even in these widely disparate species. Consistent with this hypothesis, an examination of available murine and bovine class II sequences revealed a homology in hypervariable residues between the human DRBI-1401 allele and the mouse Eps allele as well as a cow DRB allele. Consideration of these data along with intraspecies allelic sequence comparisons suggests that at least some of the interspecies similarities have emerged as the result of convergent evolution, possibly as the result of a need to react to common pathogens.The major histocompatibility complex (MHC) contains genes encoding cell-surface molecules playing a pivotal role in antigen presentation to T cells (1). The MHC of mammals is one of the most polymorphic genetic systems known, in terms of both number of alleles and structural differences between alleles. The MHC genes are divided into two classes, I and II, of which the class II genes encode two separate chains, a and A, forming a dimer on the cell surface.Almost all of the polymorphism in the class II A3 chains is concentrated in <20 amino acid residues, all of which have been hypothesized to be in direct contact with, or in close proximity to, the antigen and/or the T-cell receptor during antigen presentation (2).The MHC of swine, termed SLA (3), has been shown to exhibit extensive similarities to its human counterpart in both structure and function (4). As a large animal model for studies of transplantation biology, our laboratory has developed a herd of partially inbred miniature swine in which three independent MHC haplotypes (SLAa, SLAC, and SLAd) and three intra-SLA recombinant haplotypes (SLA, SLA", and SLAh) are maintained (5, 6). Studies of vascular grafts in these animals have shown that long-term tolerance can be obtained without exogenous immunosuppression by matching for SLA class II loci only, despite differences at class I and numerous minor loci (7). Because these results suggest the possibility of modifying transplantation immunity by genetic engineering of SLA class II genes, we have recently begun a de...