Biomedical subjects

J A Frelinger

Publications and source records attributed to J A Frelinger.

At least 73 records · Page 4Linked to original sources

PubMed1993

Cultured keratinocyte allografts fail to induce sensitization in vivo.

BACKGROUND: The use of cultured keratinocyte (CK) allografts for burn wounds offers a potentially unlimited supply of skin. It is unknown, however, whether CK allografts induce rejection in vivo. This study investigated the induction of immune responsiveness to CK allografts as measured by mixed lymphocyte response and serum cytotoxic antibody. METHODS: Female CBA mice (n = 160) were randomized to four equal groups, each receiving a 3 cm2 flank graft of autologous CBA CK (Auto CK), allogeneic C57BL/6 CK (Allo CK), C57BL/6 full thickness skin (Allo FT), or Sham. Graft take was assessed by gross and histologic examinations. Unidirectional mixed lymphocyte response was measured with graft recipient and donor splenocytes by use of tritiated thymidine uptake. Stimulation indexes were calculated. Serum cytotoxic antibody was measured by coculturing graft recipient serum with donor splenocytes and rabbit complement and assessing resultant cell killing. RESULTS: Overall graft take was 50% for Allo CK and 74% for Auto CK, Allo FT, but not Allo CK, were associated with significantly increased stimulation indexes compared with Auto CK and Sham (p < 0.01). Allo FT, but not Allo CK, resulted in elevated titers of alloantibody, reaching significant levels 10 days after grafting (p < 0.05). CONCLUSIONS: CK allografts do not result in increased in vitro T cell responses or enhanced alloantibody formation, indicating that sensitization to major histocompatibility antigens by CK does not occur. These data suggest that CK allografts may provide a possible source of grafts for victims of large burn wounds.

Animals↗

PubMed1992

LCMV-specific, class II-restricted cytotoxic T cells in beta 2-microglobulin-deficient mice.

Intracranial infection of normal mice with lymphocytic choriomeningitis virus (LCMV) causes meningitis and death mediated by CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs). beta 2-Microglobulin-deficient mice (beta 2M-/-) do not express functional MHC class I proteins and do not produce significant numbers of CD8+ T cells. When beta 2M-/- mice were infected with LCMV, many died from LCMV disease and produced a specific response to LCMV mediated by CD4+ CTLs that were class II-restricted. In these mice, CD4+ CTLs may compensate for the lack of CD8+ CTLs.

Animals↗

PubMed1992

A cross-species functional interaction between the murine major histocompatibility complex class I alpha 3 domain and human CD8 revealed by peptide-specific cytotoxic T lymphocytes.

The monomorphic cell surface glycoprotein CD8 acts as co-receptor in the recognition of peptide-major histocompatibility complex (MHC) class I complexes by cytotoxic lymphocytes (CTL) by binding to the monomorphic alpha 3 domain of the class I molecule. Positions 227 and 245 in the class I alpha 3 domain appear to be especially important for this interaction. Recent reports suggest there is no interspecies recognition between CD8 and MHC class I. In this study, hybrid genes from human class I HLA-A0201 and murine class I H-2Kb were transfected into human and mouse cells and tested in Cr-release assays using HLA-A0201-restricted influenza A matrix peptide-specific CTL as effectors. Transfected cells expressing chimeric genes comprising the alpha 1 and alpha 2 domains from HLA-A0201 together with the H-2Kb alpha 3 domain were lysed as effectively as wild-type HLA-A0201 and in both cases, killing was blocked by anti-CD8 antibody equally well. These results indicate that human CD8 can interact with the alpha 3 domain of murine class I to the same level as human class I.

Animals↗

PubMed1992

Production and characterization of two new mouse monoclonal antibodies reactive with denatured mouse class II beta chains.

We report on the production and characterization of two murine peptide specific anti-major histocompatibility complex class II chain specific monoclonal antibodies. Two new mouse monoclonal antibodies reactive with two synthetic peptides corresponding to Abb amino acids (146-157) and Abs amino acids (146-157) were produced. KL295 is the mouse anti-Abb monoclonal antibody, which reacts with denatured beta chains of H-2b, H-2d, H-2p, and H-2q, but fail to react with spleen cell lysates from H-2f, H-2j, H-2k and H-2s mice. The mouse anti Abs monoclonal antibody, KL304 in contrast reacts with the denatured Class II beta chains of H-2f, H-2j, H-2k and H-2s mice, but fails to bind H-2b, H-2d, H-2p or H-2q beta chain, suggesting residues 153-155 of this molecule to be critical for the epitope. Both KL295 and KL304 bind B10.WB (H-2j) which suggests a unique epitope in this strain of mice. Neither KL295 or KL304 react with native mouse class II cell surface molecules.

Amino Acid Sequence↗

PubMed1991

Differential expression of Q4 proteins (Qb-1) in fibroblasts and lymphocytes.

The Qb-1 protein is coded for by the Q4 gene. This gene appears to be widely transcribed in murine tissues. We have examined the subcellular localization and processing of Qb-1 in fibroblast cells. This protein has previously been reported to be secreted from activated lymphocytes. However, we report that high levels of Qb-1 are present on the surface of B10.P fibroblast cells. We also observed an unusual intracellular distribution for Qb-1. The protein appears to be highly concentrated in the endoplasmic reticulum, in addition to being on the surface. This distribution is not due to inefficient processing. The kinetics of Qb-1 processing and transport are not unusual for class I molecules. Complete resistance to endo-beta-N-acetylglucosaminidase H is acquired, indicating terminal processing. This unusual localization in fibroblast cells and the differential expression of this protein between cell types may reflect a specific role for Qb-1.

Amino Acid Sequence↗

PubMed1991

A single amino acid substitution in an MHC class I molecule allows heteroclitic recognition by lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes.

Class I molecules of the MHC bind foreign and endogenous peptides allowing recognition by the TCR on CTL. The recognition and killing of cells infected with lymphocytic choriomeningitis virus (LCMV) depends on the recognition of LCMV peptides bound to class I MHC. Mutations in class I MHC molecules have enabled the delineation of regions in the class I molecule important for binding peptides and for interaction with the TCR. We have constructed a library of class I mutants using saturation mutagenesis and report a phenotypic change resulting from a single amino acid substitution that results in the heteroclitic (increased) killing of LCMV-infected cells. This amino acid change, asparagine to serine at position 30, is in a conserved region of the class I molecule contacting the alpha 3 domain. This mutation does not result in increased expression of the class I molecule on the cell surface, does not affect the binding of CD8, and does not affect allogeneic recognition. Cold target experiments show that this heteroclitic killing is due to increased recognition by CTL. These data point toward a critical function for this region of the class I molecule in the binding of peptides or their presentation to CTL.

Animals↗

PubMed1991

A cluster of mutations in HLA-A2 alpha 2 helix abolishes peptide recognition by T cells.

In order to investigate the regions of HLA-A2 that control peptide-specific cytotoxic T lymphocyte (CTL) recognition, 37 HLA-A2 genes coding for 50 point mutations that span the alpha 2 helix were synthesized by the technique of saturation mutagenesis. Twenty-nine of these genes, which code for 41 point mutations, were transfected into C1R cells and used as targets in cytotoxicity assays, in the presence of influenza-A matrix peptide 58-68 with specific CTL as effectors. All the transfectants were recognized fully by matrix peptide-specific CTL apart from those with amino acid substitutions at positions 152, 154, 155, 156, or 161, which led to a total loss of recognition and those with mutations at residue 27 or a double mutation at 138 and 150, which were recognized in an intermediate manner. The clustering of the crucial residues that emerges may reflect direct interaction of their side-chains with peptide or the CTL receptor.

Cell Line↗

PubMed1991

Structure function analysis of the H-2 Abp gene.

The gene encoding the H-2 Ap class II beta chain was isolated from a B10.P genomic library and sequenced. This gene was also used to construct transfectants of the CH12 lymphoma clone CH12.LX, which express the Abp gene product in association with the endogenous A alpha k chain. We present here the first report of the complete nucleotide coding sequence of Abp. The predicted amino acid sequence of Abp reveals only five residues different from Abq, four of which are present in the mature peptide. These four amino acid changes could account for the differential susceptibility of H-2q vs H-2p mice to the development of collagen-induced arthritis (CIA). Antibodies specific for the transfected Abp protein induce CH12.LX cells to secrete immunoglobulin in the presence of antigen. Comparison of the amino acid sequence with other A beta chains that have been tested in signal transduction experiments suggests that amino acid 9 may be important to the signaling ability of class II A molecules.

Amino Acid Sequence↗

PubMed1991

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.

Amino Acid Sequence↗

PubMed1990

Evidence of widespread binding of HLA class I molecules to peptides.

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

Amino Acid Sequence↗

PubMed1990

The transport of class I major histocompatibility complex antigens is determined by sequences in the alpha 1 and alpha 2 protein domains.

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 X CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesized HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the alpha 1 and alpha 2 domains. Human (HLA-B7) and mouse (H-2Dp) hybrid class I genes, encoding the leader, alpha 1, and alpha 2 sequences of one species fused to the alpha 3, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, alpha 1, and alpha 2-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, alpha 1, and alpha 2 domains fused to the mouse alpha 3, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the alpha 1 and/or alpha 2 domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules.

Amino Acid Sequence↗

PubMed1990

Mutations in the alpha 1 domain of a class I gene define residues important for specific allorecognition.

Our strategy to use saturation mutagenesis to produce an unbiased collection of major histocompatibility class I mutants has resulted in unpredicted mutant phenotypes. First, we have shown data supporting our earlier work of the Dp20(Y27N) mutant. Allorecognition is altered at the clonal level while no variation in lymphocytic choriomeningitis virus (LCMV)-restricted recognition is observed. The defect does not destroy the integrity of this class I protein on the basis of three observations: (i) LCMV self-restricted recognition is not impaired, (ii) beta 2 microglobulin still associates with Dp20(Y27N) at the cell surface, and (iii) this mutant can stimulate a primary MLR. Thus, we believe Dp20(Y27N) specifically affects allorecognition, perhaps by altering self peptide associations. The Dp14(A11V;E32Q) mutant appears to interact with T cell receptors (TCR) from a cloned cytotoxic T lymphocyte, but is altered in inducing a wild type signal into the responding cell. This is presumably due to decreased interaction at the cell surface between Dp14(A11V;E32Q) and wild type-specific TCR such that variations are detected in how a cell perceives extracellular signals. Analysis of additional mutants suggests that mutant Dp163(N66S) alters the binding site for monoclonal antibodies 7-16.10 and 135, while leaving unaltered the binding site for monoclonal antibodies 34-1.2 and 11-20.3. This maps the residue responsible for 7-16.10 and 135 binding to the region of Dp163(N66S).

Animals↗

PubMed1990

Antibodies specific for Ig idiotype, but not isotype, can substitute for antigen to induce IgM secretion by a B cell clone.

Cells of the mouse B cell clone, CH12.LX, secrete IgM when cultured with nominal antigen (sheep erythrocytes, SRBC) and mAbs which bind their membrane Ek molecules. To determine whether anti-Ig antibodies can substitute for antigen in the induction of IgM secretion by CH12.LX, the B cells were cultured with anti-Ek mAbs and anti-IgM or anti-idiotype antibodies. Anti-IgM antibodies were capable of cross-linking the membrane IgM of CH12.LX, and inhibited mitogen-induced differentiation of the B cells. However, anti-IgM could not substitute for SRBC in delivering a major histocompatibility complex-restricted differentiative signal. In contrast, either polyclonal or monoclonal antibodies specific for the CH12.LX Ig idiotype were fully capable of substituting for antigen in the induction of IgM secretion by CH12.LX. The binding of anti-IgM antibodies did not prevent anti-idiotype antibodies from delivering a differentiative signal. Thus, binding of ligand to different parts of the membrane Ig molecule can result in the delivery of different biological signals to the B cell.

Animals↗

PubMed1989

Differential transport requirements of HLA and H-2 class I glycoproteins.

Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line X T-lymphoblastoid cell line (B-LCL X T-LCL) hybrid, 174 X CEM.T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or B7 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2Dp or H-2Kb class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.

Animals↗

PubMed1989

Evidence for a subpopulation of antigen-presenting cells specific for the induction of the delayed-type hypersensitivity response.

Young adult SJL mice (8 weeks of age or younger) do not mount a delayed-type hypersensitivity (DTH) response due to the failure of a macrophage antigen-presenting cell (APC) to induce TDTH effector cells. SJL mice that are 10 weeks of age or older produce a normal DTH response. This genetic defect provides a model for the investigation of functional subpopulations of APC which interact with specific subpopulations of T cells. In this study, we used this model to examine whether macrophage APC impairment involves APC-dependent immune responses other than DTH. No age-dependent differences were found in the ability of spleen cells from SJL mice to proliferate and synthesize interleukin-2 in response to concanavalin A; nor was the proliferative response to a variety of antigenic stimuli affected. In addition, no differences were observed in the contact sensitivity response or in the in vitro generation of allogeneic cytotoxic T lymphocytes (CTL). In contrast, the in vivo generation of allogeneic CTL was significantly depressed in 6-week-old SJL and could not be restored to normal by the adoptive transfer of macrophages from DTH responsive 12-week-old SJL mice. Finally, examination of the humoral response of 6-week-old SJL indicated no impairment in IgM or IgG serum antibody levels or in the induction of splenic B cells. Thus, the macrophage APC regulating the induction of TDTH effector cells does not appear to participate in the induction of T helper cells for other cellular and humoral responses. These data support the hypothesis that distinct subpopulations of APC may regulate the induction of specific immune effector mechanisms.

Animals↗

PubMed1989

Haplotype-specific differences in signaling by transfected class II molecules to a Ly-1+ B-cell clone.

CH12.LX B cells are responsive to antigen-dependent differentiative signals transmitted through their surface Ek molecules. Although CH12.LX cells express surface Ak molecules with normal protein sequence, the Ak molecules do not deliver differentiative signal to these B cells. To determine whether introduction of a new A molecule into CH12.LX cells would correct this deficiency, CH12.LX cells were transfected with the genes encoding new Aalpha and/or Abeta molecules. It was found that transfected cells responded to antigen-specific signals delivered via the Ealphak Ebetak, Aalphab Abetab, or Aalphak Abetab molecule. However, the B cells did not respond to signals generated by the molecule Aalpha kAbetak, Aalphab Abetak, Aalphak Abetad, or Aalpha kAbetau. Comparison of these sequences suggests that two Abeta residues, His-47 and Trp-197, are important to the transmission of differentiative signals to B cells.

Amino Acid Sequence↗

PubMed1989

Diminished expression of the T cell receptor on the expanded lymphocyte population in MRL/Mp-lpr/lpr mice.

MRL mice homozygous for the recessive lpr gene develop an accelerated autoimmune syndrome and massive lymphadenopathy. Because the function of the expanded lymph node population is unclear, we have studied the subunits of the T cell receptor for antigen (TcR). DNA and RNA were prepared from MRL/Mp-lpr/lpr (lpr) and congenic MRL/Mp(-)+/+ (+/+) mice by standard techniques and studied by Southern blot, northern blot, and dot blot analysis using the cDNAs TT11, specific for the TcR alpha chain; 86T5, specific for the TcR beta chain; and T3 delta; specific for the subunit of the T3 molecule. Surface protein was immunoprecipitated with antisera 8177, which recognizes TcR framework determinants, and resolved by diagonal SDS-PAGE. FACS analysis was performed with a monoclonal antibody to murine T3, and with the KJ16-133 and F23.1 monoclonal antibodies, which recognize determinants encoded by the V beta 8 subfamily of beta chain variable region genes. When compared with +/+ controls, surface TcR density as detected by immunofluorescence using all three antibodies was significantly diminished on lpr spleen and lymph node cells, as well as on lpr lymph node cells which had been depleted of L3T4+ and Ly2+ cells by negative selection. There appeared, however, to be selective expression of the genes encoding the epitopes binding F23.1. Southern blot analysis of DNA showed polyclonal rearrangements of the TcR beta chain genes. There were increased alpha, beta, and T3 delta RNA transcripts in the double negative lymph node cells. The paradoxical decrease in TcR surface expression in the setting of large quantities of full length transcript is yet to be explained.

Animals↗

PubMed1988

Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells.

Amino Acid Sequence↗