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J A Frelinger

Publications and source records attributed to J A Frelinger.

At least 91 records · Page 5Linked to original sources
PubMed1988

Signaling to a B-cell clone by Ek, but not Ak, does not reflect alteration of Ak genes.

The mouse B-cell clone, CH12.LX (Iak, Ly-1+, mu+, delta+), can be induced to differentiate and secrete antibody in an antigen-specific, H-2-restricted manner. Induction requires two signals. One must be provided by the binding of specific antigen to the membrane IgM; the other is delivered by the binding of Ek-specific T-cell hybridomas to the Ek molecules of CH12.LX (Bishop and Haughton 1986). Previous studies demonstrated that Ek-specific monoclonal antibodies (mAbs) could substitute for T cells in delivering the second differentiative signal (Bishop and Haughton 1986). Although CH12.LX cells present Ak to Ak-restricted or alloreactive T-helper cells, neither T cells nor mAbs specific for Ak induce differentiation (Bishop and Haughton 1986). However, since the Akspecific mAbs tested previously were beta-chain-specific and the Ia epitope specificity of the T cells used was unknown, it is possible that the differentiative signal delivered to the CH12.LX class II molecule is chain-specific. Here we report the effects of ten additional Iak-specific mAbs upon the differentiation of CH12.LX. In addition, a cDNA library was prepared from CH12.LX cells, clones corresponding to the alpha and beta chains of the Ak molecule were isolated, and their nucleotide sequences were determined. Finally, the Ak and Ek molecules of CH12.LX and H-2k spleen cells were compared by two-dimensional gel electrophoresis to examine possible post-translational differences in the Iak molecules of CH12.LX.

Amino Acid Sequence↗
PubMed1988

Saturation mutagenesis of a major histocompatibility complex protein domain: identification of a single conserved amino acid important for allorecognition.

The interactive association between T lymphocytes and their target cells is an important system of cell-cell interactions. Major histocompatibility complex class I molecules are the cell surface structures recognized by cytolytic T lymphocytes. To define the molecular structures recognized by cytotoxic T lymphocytes, we have saturated the 270-base-pair alpha 1 exon of the H-2Dp gene with point mutations, rapidly producing a "library" of 2.5 x 10(3) independent mutants. The library contains enough recombinant clones (each clone encoding approximately one amino acid replacement mutation) to predict a mutation at each nucleotide position of the alpha 1 exon. The functional analysis of the first five transfected gene products tested has shown that mutation of a conserved tyrosine at position 27 to asparagine destroys recognition of the H-2Dp gene product by polyclonal alloreactive cytotoxic T lymphocytes. Recognition of the same mutant molecule by three monoclonal antibodies and H-2-restricted lymphocytic choriomenengitis virus-specific cytotoxic T lymphocytes is unaffected.

Amino Acid Sequence↗
PubMed1988

High-level expression of a bioengineered, cysteine-free hepatocyte-stimulating factor (interleukin 6)-like protein.

Hepatocyte-stimulating factor, interferon-beta 2, B-cell stimulation factor 2, and hybridoma/plasmacytoma growth factor are identical proteins presently referred to as interleukin 6 (IL-6). Through the use of synthetic oligonucleotide technology, we have constructed a biologically active recombinant IL-6 (rIL-6) gene based on the sequence of a human IL-6 cDNA. The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein that is expressed at high levels in Escherichia coli as a tripartite fusion protein. Cleavage of the fusion protein with collagenase releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity. We show that the rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (i) protect cells from viral infection, (ii) stimulate the synthesis of fibrinogen in rat FAZA 967 cells, and (iii) induce the terminal differentiation of B cells, resulting in elevated secretion of immunoglobulin.

Amino Acid Sequence↗
PubMed1988

Mouse genetic locus Lps influences susceptibility to Neisseria meningitidis infection.

We surveyed a number of inbred mouse strains for susceptibility to meningococcemia. Mice of all strains became bacteremic after intraperitoneal injection of a serogroup C, serotype 2a human disease isolate, but the strains differed in levels of bacteremia, indicating influences of the host genome on susceptibility. There was no significant correlation between level of bacteremia and differences at major histocompatibility or immunoglobulin loci; the Salmonella susceptibility locus, Ity; the complement C5 locus, Hc; the antibody response locus, xid; or the transferrin locus, Trf. However, the Lps locus, which influences a range of host cellular responses to endotoxin and affects susceptibility to Salmonella typhimurium, did influence susceptibility to meningococcemia. There were significant differences in levels of bacteremia between C3H/HeJ (Lpsd) mice and each of the other strains (all Lpsn). We confirmed the association of the Lpsd genotype with susceptibility by using coisogenic strains from two widely separated mouse lineages: C3H and B10. Lpsd mice experienced a 1,000-fold proliferation of bacteria and were bacteremic for days before clearing the infection. In contrast, Lpsn mice cleared the bacteremia in less than 1 day. There was no difference in meningococcal growth in vitro in serum from C3H/HeJ and coisogenic C3H/HeN (Lpsn) mice, suggesting that the Lps-related difference in susceptibility may involve a cellular response.

Animals↗
PubMed1988

Southern blot analysis of major histocompatibility genes of lpr mice.

Several recent functional and immunofluorescent studies have suggested that abnormal major histocompatibility genes may be expressed in mice homozygous for the autoimmune disease-accelerating lpr (lymphoproliferation) gene. In an effort to establish the molecular basis of the expression of abnormal MHC molecules in these mice, we used MHC class I- and class II-specific cDNAs to probe endonuclease-digested genomic DNA from strains expressing lpr to look for restriction fragment length polymorphisms (RFLPs). The RFLP pattern of MRL/Mp-lpr/lpr (MRL/lpr) DNA, as determined using five restriction enzymes, was identical to that of MRL/MP-+/+ and typical of the H-2k haplotype exhibited by C3H/HeSnJ and AKR/J, which are the two H-2k haplotype strains from which MRL was derived. DNA from C57BL/6-lpr/lpr (B6/lpr) exhibited a pattern similar to C57BL/6, again indicating that the lpr gene did not alter MHC genes, within the limitations of this approach. Because macrophages derived from lpr mice had been specifically reported to express altered MHC haplotypes, DNA was prepared from MRL/lpr peritoneal macrophages and Southern blotting was performed using probes for I-A beta and I-E beta. The patterns observed were typical of H-2k DNA. The data thus indicate that the abnormalities of MHC genes previously observed in lpr mice may be due to the influence of other genes on MHC gene products, but are probably not due to alterations in MHC genes themselves.

Animals↗
PubMed1987

Widespread transcription of a Qa region gene in adult mice.

The mouse MHC class I family includes genes encoded in four regions: H-2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene.

Animals↗
PubMed1987

New family of exon-shuffled recombinant genes reveals extensive interdomain interactions in class I histocompatibility antigens and identifies residues involved.

The specificities of an extensive panel of anti-H-2Dd monoclonal antibodies, which had been previously characterized using exon-shuffled H-2Dd/H-2Ld molecules and a number of anti-H-2DP antibodies, were examined using H-2Dd/H-2DP recombinants. The use of this new family of recombinant antigens revealed extensive interaction between the membrane-distal (N and Cl) domains of class I molecules. 20 out of 48 mAbs recognize complex epitopes formed by the interaction of these two domains. These antibodies exhibit a number of distinct patterns of crossreactivity with other class I proteins, revealing the presence of multiple epitopes within the region of domain interaction. Comparison of the data presented here with those from previous work allowed the identification of a small number of residues in the Cl domain that participate in the generation of complex epitopes involving both the N and Cl domains. The results are discussed in terms of the structural information available for these two domains.

Amino Acid Sequence↗
PubMed1987

The alpha 1 and alpha 2 domains of H-2 class I molecules interact to form unique epitopes.

Mouse class I antigens are the major targets of cytolytic T lymphocytes in both major histocompatibility complex (MHC)-restricted and allogeneic responses. Considerable evidence has recently accumulated demonstrating that MHC class I molecules encoded by genes whose alpha 1 and alpha 2 coding exons were interchanged are not recognized by T lymphocytes specific for parental class I products. Along with the loss of T-cell reactivity, there is a loss of recognition by some, but not all monoclonal antibodies. In this communication we report that the loss of reactivity by monoclonal antibodies is accompanied by the gain of new epitopes caused by the interaction of alpha 1 and alpha 2 domains. These epitopes are immunodominant. They are the major determinant recognized by polyclonal antisera raised by immunization with L cells transfected with exon-shuffled class I genes. Four new monoclonal antibodies have been produced which recognize at least two separate epitopes caused by the interaction of the alpha 1p and alpha 2d domains.

Animals↗
PubMed1987

The expression and detection of MHC class I antigens on murine neuroblastoma and ependymoblastoma lines.

It has been reported that human neuroblastoma lines are almost devoid of class I transplantation antigens, while human glioma lines express these antigens. Other studies have also shown a paucity of class I antigens on the murine neuroblastoma line N2A, and the expression of these antigens by the murine ependymoblastoma G26 lines. Such differences might represent heterogeneity in class I antigen expression by different brain cell types, and the importance of this to the immunology of the brain prompted us to re-examine class I expression by these cell lines in more detail. Using an exhaustive number of approaches, we were not able to detect significant differences in class I surface antigen expression between N2A and the G26 lines. We compared the murine neuroblastoma line Cl300 and its cloned derivative, N2A, to the lines G26-20 and G26-24. Antibody-dependent, complement-mediated cytotoxicity revealed detectable levels of both K and D region antigens on these lines. Immunocytofluorometric analysis further confirmed that these lines express high levels of class I antigens, although due to their large sizes, the surface densities of class I antigens on these cells are lower than splenocytes. This lower density of class I molecules did not impede the capacity of either the neuroblastoma or the G26 lines to serve as targets of H-2K- or D-specific T effectors. Finally, comparison of these two cell types for class I RNA transcripts also revealed no difference. Thus, our findings which are the most detailed study of these lines are drastically different from findings in humans as well as earlier findings in the murine system. Likely explanations are discussed and precautions are given for the study of class I antigen expression by these lines.

Animals↗
PubMed1986

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene.

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.

Amino Acid Sequence↗
PubMed1986

Induction of antigen presentation ability in purified cultures of astroglia by interferon-gamma.

The role of the immune system in the central nervous system has been elusive. Our original description of Ia bearing cells in the central nervous system was controversial, although it has now been confirmed in a variety of systems in both mouse and humans. The function of Ia bearing cells is however still unclear. Recently, others have shown that astrocytes from rats with EAE could present myelin basic protein to T cell clones; however, no other antigens were tested. We have used the culture systems of McCarthy and DeVellis to produce purified cultures of astrocytes and oligodendroglial cells from newborn mouse brains. Newborn brains were chosen since it is impossible to obtain pure cultures of differentiated brain cells from adult mice. Using these cultures, we showed that astrocyte, but not oligodendrocyte cultures treated with ConA supernatants or recombinant IFN-gamma are able to present antigen to appropriate but not inappropriate T cell hybrids. Untreated cells of either the astrocyte or oligodendroglial cell populations were ineffective at antigen presentation. Concomitant with this increase in antigen presenting ability, follows an increase in both the number and density of MHC class I and class II antigens. Antigen presentation was inhibited by appropriate but not inappropriate anti Ia monoclonal antibodies. Anti class I antibodies were ineffective. Depletion experiments showed that both I-A and I-E molecules are expressed on the antigen presenting cells. Thus, we have been able to show that Ia+ cells derived from pure cultures of astrocytes are able, after induction with IFN-gamma, to present antigen to T cell hybrids. This suggests a possible physiologic role of Ia bearing cells in CNS in initiation of immune responses.

Animals↗
PubMed1985

Response of glioma cells to interferon-gamma: increase in class II RNA, protein and mixed lymphocyte reaction-stimulating ability.

Previous results by ourselves and others demonstrated that brain cells and cell lines express major histocompatibility complex class II antigens. We examined interferon-gamma (IFN-gamma)-mediated induction of human class II antigen expression on the glioma cells. Purified IFN-gamma induced the expression of HLA-DR antigens on the surface of the glioma cell lines U-373 MG and U-105 MG. Concomitant increase of HLA-DR alpha- and HLA-DC beta-specific RNA in the cytoplasm was also observed after treatment with IFN-gamma. Increases of class II antigen paralleled the increased level of class II-specific RNA. The effect of IFN-gamma on the induction of human class II antigen expression was dose and time dependent. A marked induction of human class II antigen expression was observed when glioma cells were cultured with more than 100 U/ml of IFN-gamma. Little or no induction was observed with less than 50 U/ml of IFN-gamma. Compared to human blood monocytes, glioma cells needed higher concentrations of IFN-gamma for the induction of class II antigen expression. In allogenic mixed lymphocyte cultures, the glioma cell line U-373 MG stimulated a mixed lymphocyte response (MLR). MLR-stimulating capacity was augmented by IFN-gamma. The concomitant augmentation of class II antigen levels and MLR-stimulating capacity suggests that the most relevant factor for MLR stimulation may be antigen density. This is the first report of MLR stimulation by a glioma cell line.

Dose-Response Relationship, Immunologic↗
PubMed1985

The defect in delayed-type hypersensitivity of young adult SJL mice is due to a lack of functional antigen-presenting cells.

SJL mice exhibit a strain-specific age-dependent delay in the maturation of delayed-type hypersensitivity (DTH) responsiveness. They do not attain "adult" levels of DTH responsiveness until the 10th week of age, which is 4 to 6 weeks later than the other strains of mice tested. In this report we demonstrate that spleen cells, resident peritoneal cells and thioglycollate-elicited peritoneal exudate cells are all able to transfer DTH responsiveness from naive 12-week-old DTH responders to 6-week-old nonresponders. Transfer prior to immunization was more efficient at eliciting a response than transfer after immunization. As few as 5 X 10(4) cells from 12-week-old SJL mice can adoptively transfer responsiveness to unresponsive 6-week-old animals. The active cell was found to be adherent, radiation (2000 rds) resistant, I-A+, Thy-1- and Mac-1+. I-A compatibility between the adoptively transferred population and the nonresponder mice is required. These data suggest that young adult SJL mice lack a functional population of antigen-presenting cells specific for DTH and that the appearance of these cells is under maturational control.

Aging↗
PubMed1985

Expression in L cells of transfected class I genes from the mouse major histocompatibility complex.

One of the major surprises of the molecular analysis of major histocompatibility complex (MHC) genes is the large number of class I (K/D)-related sequences in the genome. Both restriction fragment length polymorphisms and cosmid cloning experiments showed them all to be closely linked to the MHC. Until now little information was available concerning either their expression or recognition by the immune system. Here we report that these non-K/D genes can provoke antibody responses and be recognized by cytolytic T cells. Immunization of C3H mice with L cells transfected with class I genomic clones resulted in antisera that reacted preferentially with cells from strain B10.P (the gene donor). Thus, these genes can be expressed by L cells. These products were recognized by cytolytic T cells produced by mixed lymphocyte culture with B10.P stimulators. One gene, represented in clone lambda 3a, was chosen for further analysis. A restriction fragment length polymorphism, detected between B10.P (KpDp) and B10.F(14R) (KbDp) and between B10 (KbDb) and B10.F(13R) (KpDb), has enabled us to map the lambda 3a sequence to the D or Tla region. Restriction endonuclease mapping of the lambda 3a clone shows that the gene is intact and that, although many restriction sites are conserved, the gene in lambda 3a differs from other class I genes. When the lambda 3a clone was transfected into mouse L cells, a new product was expressed. Cells expressing this product (designated L3a cells) were killed by primary D-end-reactive, allospecific cytolytic T lymphocytes. The L3a cells were unreactive with monoclonal antibodies specific for the Kp,Dp,Qa-2, Tla.3, and Tla.5 molecules.

Animals↗