muramidase and Lymphoma--B-Cell

Topics: Amino Acid Sequence; Animals; Histocompatibility Antigens Class II; Humans; Lymphoma, B-Cell; Molecular Sequence Data; Muramidase; Peptide Fragments; Protein Binding; Tumor Cells, Cultured

The purpose of this study was to describe patients initially carrying a diagnosis of primary intraocular lymphoma who were ultimately diagnosed with ocular sarcoidosis.. The medical records of patients evaluated between 1995 and 2007 fitting the criteria described earlier were identified, and pertinent clinical findings allowing for the diagnosis of sarcoidosis are described.. Nine patients between the ages of 52 and 83 were referred with a diagnosis of primary intraocular lymphoma but were ultimately diagnosed with sarcoidosis. The most common clinical signs found in these patients that are atypical for primary intraocular lymphoma but common in sarcoidosis were multifocal choroiditis (n = 7) and cystoid macular edema (n = 6). Additional findings included keratic precipitates, posterior synechiae, and Koeppe nodules. Chest computerized tomography was consistent with sarcoidosis in seven of eight tested patients, and five of these patients had normal chest x-rays. Other findings included elevated angiotensin-converting enzyme and/or lysozyme, and biopsy revealing noncaseating granulomas.. Although primary intraocular lymphoma should always be in the differential diagnosis of older patients who present with signs of ocular inflammation, ophthalmologists must also consider other etiologies, including sarcoidosis. A chest computerized tomography may be helpful in the diagnosis, particularly when laboratory findings are supportive of sarcoidosis.

Topics: Aged; Aged, 80 and over; Diagnosis, Differential; Diagnostic Errors; Eye Diseases; Eye Neoplasms; Female; Humans; Lymphoma, B-Cell; Magnetic Resonance Imaging; Male; Middle Aged; Muramidase; Peptidyl-Dipeptidase A; Sarcoidosis; Tomography, X-Ray Computed; Uveitis; Vitreous Body

The processing by antigen-presenting cells (APC) of the protein hen egg-white lysozyme (HEL) results in the selection of a number of peptide families by the class II major histocompatibility complex (MHC) molecule, I-A(k). Some of these families are expressed in very small amounts, in the order of a few picomoles/10(9) APC. We detected these peptides from an extract of class II MHC molecules by using monoclonal anti-peptide antibodies to capture the MHC-bound peptides prior to their examination by HPLC tandem mass spectrometry. Here, we have identified several members of a family of peptides encompassing residues 20-35, which represent less than 1% of the total HEL peptides. Binding analysis indicated that the core segment of the family was represented by residues 24-32 (SLGNWVCAA). Asn-27 (shown in boldface) is the main MHC-binding residue, mapped as interacting with the P4 pocket of the I-A(k) molecule. Analysis of several T cell hybridomas indicated that three residues contacted the T cell receptor: Tyr-23 (P-1), Leu-25 (P3), and Trp-28 (P5). The HEL peptides isolated from the APC extract were sulfated on Tyr-23, but further analysis showed that this modification did not occur physiologically but took place during the peptide isolation.

Topics: Alanine; Amino Acid Sequence; Animals; Binding Sites; Chickens; Epitopes; Female; Histocompatibility Antigens Class II; Kinetics; Lymphoma, B-Cell; Mice; Muramidase; Peptide Fragments; Phosphorylation; Sulfates; Tumor Cells, Cultured

Knowing the abundance of peptides presented by MHC molecules is a crucial aspect for understanding T cell activation and tolerance. In this report we determined the relative abundance of four distinct peptide families after the processing of the model Ag hen egg-white lysozyme. The development of a sensitive immunochemical approach reported here made it possible to directly quantitate the abundance of these four epitopes presented by APCs, both in vitro and in vivo. We observed a wide range of presentation among these four different epitopes presented on the surface of APCs, with 250-fold differences or more between the most abundant epitope (48-63) and the least abundant epitopes. Importantly, we observe similar ratios of presentation from APCs in vitro as well as from APCs from the spleens and thymi of hen egg-white lysozyme transgenic mice. We discuss the relationship between the amount of peptide presented and their binding to I-A(k) molecules, immunogenicity, and tolerogenicity.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; Antigen-Presenting Cells; Chickens; Enzyme-Linked Immunosorbent Assay; Histocompatibility Antigens Class II; Immunodominant Epitopes; Lymphoma, B-Cell; Mice; Mice, Transgenic; Molecular Sequence Data; Muramidase; Peptide Fragments; Protein Binding; Spleen; Thymus Gland; Tumor Cells, Cultured

The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Antigen Presentation; Histocompatibility Antigens Class II; Immunodominant Epitopes; Lymphoma, B-Cell; Mice; Molecular Sequence Data; Multigene Family; Muramidase; Mutagenesis, Site-Directed; Peptide Fragments; Proline; Tumor Cells, Cultured

Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their alpha- or beta-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak beta-chain is required for the presentation of the hen egg lysozyme determinant (46-61) following BCR internalization, but that dependence is not seen for the (34-45) determinant derived from the same protein. The tail of the beta-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II beta-chains control the loading of determinants produced after extensive Ag processing.

Topics: Animals; Antigen Presentation; Cell Line; Cycloheximide; Cytoplasm; Egg Proteins; Histocompatibility Antigens Class II; Intracellular Fluid; Liposomes; Lymphoma, B-Cell; Mice; Mice, Inbred CBA; Mice, Transgenic; Muramidase; Mutation; Peptide Fragments; Protein Binding; Receptors, Antigen, B-Cell

Specific antibodies increase antigen uptake and presentation by antigen-presenting cells via the B cell receptor in B cells or FcgammaR in dendritic cells. To determine whether the interaction between antibody and antigen could influence the set of peptides presented by MHC II molecules, we analyzed the presentation of different CD4(+) T cell epitopes of hen egg-white lysozyme (HEL) after the capture of immune complexes formed between HEL and seven different specific mAb. The 103-117 T cell epitope (I-E(d)) was specifically and selectively up-regulated by the D1.3 and F9.13.7 mAb that binds to proximal loops in the native structure of HEL. Furthermore, Ii-independent T cell epitopes exposed on the HEL surface (116-129 and 34-45, I-A(k) restricted) which require a mild processing involving the recycling of MHC II molecules were selectively up-regulated by mAb that overlap those T cell epitopes (D1.3 and D44.1). However, F10.6.6, somatically derived from the same germ line genes as D44.1 and exhibiting an higher affinity for HEL, was without effect on the presentation of the 34-45 epitope. An Ii-dependent T cell epitope buried into the tertiary structure of HEL (45-61, I-A(k) restricted) and requiring the neosynthesis of MHC II was up-regulated by high-affinity mAb recognizing epitopes located at the N- or C-terminus of the T cell epitope. These results strongly suggest that (i) the spatial relationship linking the T cell epitope and the B cell epitope recognized by the mAb, (ii) the intrinsic processing requirements of the T cell epitope, and (iii) the antibody affinity influences the presentation of a given T cell epitope.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigen Presentation; Antigen-Antibody Complex; Antigen-Presenting Cells; CD4-Positive T-Lymphocytes; Chickens; Egg White; Epitopes; Flow Cytometry; Histocompatibility Antigens Class II; Hybridomas; Lymphoma, B-Cell; Mice; Muramidase; Receptors, Fc; Tumor Cells, Cultured

Activation of CD4+ T cells requires processing of exogenous protein antigens by antigen-presenting cells (APC). A macrophage hybridoma and B cell lymphoma were comparable in their ability to process hen egg lysozyme (HEL), which involves reduction of its disulfide bonds. The intracellular levels of cysteine and glutathione, major physiological thiols, based on protein content were similar within these cell lines. In addition, the cysteine transport pathway in viable cells was assessed by 35S-cystine uptake. For macrophages, the majority of the radioactivity resided in high density subcellular fractions of Percoll gradients that comigrated with lysosomal beta-galactosidase (beta-gal). Besides the lysosomes, low density fractions cosedimenting with endosomes incorporated the radiolabel in the B cells. Both peaks of radioactivity disappeared when the B cells were incubated with unlabeled carboxymethyl-cysteine (CM-cysteine), a specific competitor of the plasma membrane CG transport system. The distinct gradient profiles of radiolabel uptake in the cells correlated with a difference in their capacity to process the transferrin-lysozyme conjugate (TF-HEL). TF-HEL was significantly more stimulatory than HEL in inducing a HEL-specific T cell response with the B cells as the APC. However, the potencies of TF-HEL and HEL were similar when the macrophages were the APC. Thus, the intracellular location of cysteine transport activity may be cell lineage-dependent, and its presence may, in part, determine whether an organelle is a productive site of processing antigens with disulfide bonds that is necessary for CD4+ cell activation.

Topics: Antigen Presentation; B-Lymphocytes; beta-Galactosidase; Biological Transport; Carbocysteine; Cysteine; Cystine; Disulfides; Endosomes; Glutamine; Glutathione; Hybridomas; Lymphoma, B-Cell; Lysosomes; Macrophages; Muramidase; Oxidation-Reduction; Sulfhydryl Compounds; Transferrin; Tumor Cells, Cultured

We have previously identified an intracellular compartment involved in the association between processed lysozyme and IAk major histocompatibility complex class II molecules (called the lysozyme-loading compartment (LLC)). Here, we show that the LLC polypeptide composition analyzed by two-dimensional gel electrophoresis shares similarities with that of early endosomes, but not with that of late endosomes. The transferrin receptor, a well known marker for both early and recycling endosomes, colocalizes with IAk molecules in LLC. Moreover, both transferrin and fluid-phase markers have access to LLC after 15 min of internalization. In the presence of concanamycin B, SDS-stable dimer formation and transport of class II molecules out of LLC are impaired. In contrast, nocodazole treatment has no effect. These results suggest that LLC is a specialized compartment of the recycling pathway involved in lysozyme loading and in the targeting of lysozyme-major histocompatibility class II complexes toward the cell surface.

Topics: Animals; Anti-Bacterial Agents; B-Lymphocytes; Cell Fractionation; Dimerization; Electrophoresis, Gel, Two-Dimensional; Endocytosis; Endosomes; Histocompatibility Antigens Class II; Horseradish Peroxidase; Kinetics; Lymphoma, B-Cell; Macrolides; Mice; Microscopy, Electron; Muramidase; Nocodazole; Tumor Cells, Cultured

The processing of exogenous antigens and the association of peptides with class II molecules both occur within the endocytic pathway. 2A4 B lymphoma cells of the H-2k haplotype were grown in the presence or the absence of two different exogenous antigens (hen egg lysozyme and ribonuclease A) internalized by fluid-phase endocytosis. Using subcellular fractionation techniques, we demonstrate that, in the presence of hen egg lysozyme, newly synthesized SDS-stable class II molecules are detected in a dense endocytic compartment which does not have the characteristics of neither early and late endosomes nor lysosomes. In contrast, no SDS-stable class II molecules are observed between ribonuclease A and newly synthesized class II molecules. Interestingly, when class II molecules are analyzed at steady state, SDS-stable class II molecules induced by ribonuclease A are found in a compartment cosedimenting with late endosomes. These results suggest that the tight associations between ribonuclease A or hen egg lysozyme with class II molecules occur in distinct endocytic compartments and that these associations may depend on the sensitivity of antigens to proteolysis.

Topics: Animals; Antigen Presentation; B-Lymphocytes; Biological Transport; Cell Compartmentation; Chickens; Endocytosis; Histocompatibility Antigens Class II; Lymphoma, B-Cell; Mice; Muramidase; Ribonuclease, Pancreatic; Subcellular Fractions; Tumor Cells, Cultured

We describe here the G12 pro-B cell clone that has been isolated from an IL-7 transgenic mouse. This clone has the phenotype B220+, BP-1+, HSA+, CD43+, lambda5+, and CD25-, and has its Ig locus in a germline configuration. G12 cells spontaneously express cell-surface MHC class II molecules, although to a much lesser extent than the mature M12.4.1 B-cell lymphoma. G12 cells can process and present the native Hen Egg Lysozyme (HEL) to an MHC class II-restricted T-cell hybridoma. The efficiency of presentation is inferior to that obtained with M12.4.1 cells. This is the first report where a pro-B cell can serve as APC in an MHC class II-restricted presentation.

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; B-Lymphocytes; Cell Differentiation; Cell Line; Clone Cells; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Histocompatibility Antigens Class II; Lymphoma, B-Cell; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Muramidase; Peptides; Protein Binding; Stem Cells; Transcription, Genetic; Tumor Cells, Cultured

LPS is the most important antigen of Brucella bacteria which are gram-negative facultative intracellular pathogens infecting a large proportion of animals and humans in the world. In order to get insights into the immune response mechanisms monitored by Brucella, its LPS was used as a model antigen. S-LPS, R-LPS, lipid A and O-chain purified from Brucella abortus were tested in their capacity of inducing SDS-resistant MHC class II molecules after incubation with murine B lymphoma cells. S-LPS and O-chain gave a significant response suggesting that O-chain might induce an association with class II itself or might act as a carrier for antigens to bind MHC class II molecules.

Topics: Animals; Brucella abortus; CD4-Positive T-Lymphocytes; Chickens; Drug Resistance; Female; Histocompatibility Antigens Class II; Hot Temperature; Immunosorbent Techniques; Lipid A; Lipopolysaccharides; Lymphoma, B-Cell; Mice; Mice, Inbred CBA; Muramidase; Sodium Dodecyl Sulfate; T-Lymphocytes, Helper-Inducer; Tumor Cells, Cultured

We have examined the interactions of various peptides with the mouse class II major histocompatibility complex molecule I-Ak. The peptides were derived from the model protein hen egg white lysozyme (HEL). The immunodominant peptide of HEL is a 10-mer, residues 52-61. Our previous work established that this sequence contains the key residues for binding and presentation to T cells. Now we show that the binding of this 10-mer sequence resulted in complexes of I-Ak and peptide that, in SDS/PAGE (without boiling the protein), rapidly dissociated from the component alpha and beta chains. The binding interactions were studied in vitro, by incubating purified I-Ak and radiolabeled peptide, or ex vivo, by using antigen-presenting cells incubated with peptides. Peptides with additional residues at either the amino or carboxyl terminus behaved dramatically differently. Complexes of I-Ak with the longer peptides were stable to SDS/PAGE. Very few amino acid additions result in the change from unstable to stable complexes. The important issue here is that when cultured with HEL, antigen-presenting cells selected the HEL peptides containing the 52-61 sequences that favored stability [Nelson, C. A., Roof, R. W., McCourt, D. W. & Unanue, E. R. (1992) Proc. Natl., Acad. Sci. USA 89, 7380-7383]. Also, from other studies, such sequences correlate with a high immunogenicity of the peptide. We conclude that there are structural features of peptides that change the stability of the class II molecule and that are independent of the "core" peptide seen by the T cells.

Topics: Amino Acid Sequence; Animals; Binding Sites; Chickens; Histocompatibility Antigens Class II; Lymphoma, B-Cell; Mice; Molecular Sequence Data; Muramidase; Peptide Fragments; T-Lymphocytes