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Abstract: A strain ofAlternaria alternata (Fr.) Keissl, when grown on wheat bran Czapek Dox medium was found to secrete one neutral and two alkaline proteases. The purified enzymes were found to be endo peptidases, the alkaline proteases being serine proteases and neutral proteases being cysteine proteases. Fructose when added to the culture medium was found to give rise to a new neutral protease at the expense of the neutral protease produced in the absence of fructose and was also found to enhance the production of alkaline proteases. It also appears that fructose modifies the alkaline proteases with respect to some characteristics such asV max, Ea etc. Sodium dodecyl sulphate Polyacrylamide gel electrophoresis indicated a significantly altered protein profile in fructose supplemented medium.

Ia molecules play a key role in antigen recognition by T lymphocytes. To analyze the structural features of the individual alpha and beta chains relevant to the assembly of intact Ia molecules, mouse fibroblasts were cotransfected with various combinations of haplotype- and isotype-mismatched Ia alpha/beta gene pairs. Two important points emerged. First, the level of surface expression of a given haplotype-mismatched A alpha A beta pair appears to depend upon the alpha and beta chain alleles involved. Second, transfection with some isotype-mismatched combinations such as Ed alpha Ad beta results in a significant level of surface expression of a stable mixed-isotype dimer, which also appears to be normally expressed at a low level by an Iad-positive B lymphoma. Moreover, a T-cell hybridoma specific for human gamma globulin and restricted by the Ed molecule was found to be efficiently stimulated by the Ed alpha Ad beta-positive transfectant in the presence of antigen. The stimulation was specifically inhibited by monoclonal antibodies directed to either the Ia or the L3T4 molecule. These findings suggest that the estimates of the potential number of Ia molecules available in an animal for restricting T-lymphocyte recognition of antigens must be revised.

We studied the antigen-presenting capacity of mouse L fibroblasts transfected with genes encoding Ia polypeptides of the major histocompatibility complex (MHC). These cells function as efficient antigen-presenting cells (APC) in stimulating peptide antigen-specific MHC-restricted proliferation of long-term T-cell lines, thus establishing the capacity of Ia-expressing L-cell transfectants to present antigens to apparently normal T cells. However, in contrast to splenic APC, L-cell transfectants fail to present native hen egg-white lysozyme to the same T cells. Since this result is similar to that obtained with physiologic APC pretreated to prevent antigen degradation, it suggests that L-cell transfectants, without such pretreatments, may be compromised in their ability to process native lysozyme. However, since such transfectant cells have been shown to present other complex polypeptides such as keyhole limpet hemocyanin, a random copolymer of glutamic acid, alanine, and tyrosine, and influenza virus neuraminidase, this observation suggests that protein antigens differ in the stringency of processing requirements.

Using synthetic peptides as antigens, it was found that T cell clones of a given haplotype specific for 13-16 amino acid peptides could be clearly distinguished by the varied influence of amino acid substitutions on recognition. This was true for different antigenic determinants within peptides 81-96 and 74-86 of hen egg-white lysozyme, recognized in the context of the I-Ab and I-Ak molecules, respectively. Considerable complexity was demonstrated in the induced T cell repertoire specific for apparently single determinants, which implies that diversity of T cell recognition approaches that for B cells. The implications of the degeneracy of T cell recognition are discussed in the context of mechanisms through which Ia molecules restrict recognition and theories of Ir gene defects.

Self or foreign cellular proteins provide peptides for presentation by major histocompatibility complex (MHC) class I molecules on the surface of antigen presenting cells (APC). Surprisingly, several studies have shown that T-cells can recognize APC transfected with antigen genes that were not present in the appropriate translational context. To understand the basis of this phenomenon, APC were transfected with DNA constructs encoding the OVA257-264 (SL8) peptide, but with varying translation initiation codons. We report that, in addition to ATG, 6 other codons (ATT, ACG, CTG, GCG, TGG, GAT) also allowed presentation to SL8-Kb-specific T-cells. Significantly, this set includes 3 of 4 known non-ATG translation initiation codons strongly suggesting that cryptic translation accounts for this phenomenon. Although expression of the SL8-Kb complex was readily detected by T-cell activation, the amount of processed peptides was below detection limit (< 30 copies/cell) in cell extracts. Thus, the fortuitous presence of these cryptic translation initiation sites in transcribed genes can explain how peptide MHC complexes were obtained in sufficient amounts for T-cell activation. The translation initiation codons identified here could also be useful for identifying potential open reading frames that possess biological and/or immunological activities.

Ak- or Ek-restricted T cells, generated by immunization with a 23-amino-acid peptide of hen egg-white lysozyme (amino acid 74-96), showed a strict correlation between the minimal peptide determinant recognized and the Ia molecule restricting recognition. All Ak-restricted clones obtained from six independently derived lines recognized determinants contained within peptides 74-86, while Ek-restricted clones recognized determinants within 85-96. This correlation was true whether B10.A mice (Ak, Ek) were immunized with peptide 74-96 or with each of the two smaller peptides (74-86 or 85-96). Furthermore, a T cell response could be obtained to peptide 74-86, but not to peptide 85-96 in B10.A (4R) mice, which express only the Ak molecule. Thus, an Ia molecule-associated selectivity exists in the choice of T cell determinants even within this small 23-amino-acid peptide antigen. Significant differences were noted, however, in the boundaries of the minimal peptide determinants recognized within peptide 74-96 by Ak- or Ek-restricted T cells, in comparison to those recognized by Ab-restricted T cells. These results indicate that interaction of the same peptide with distinct Ia molecules results in recognition of unique aspects of the antigenic determinants by the T cell receptor.

The antigen presentation pathway yields peptide-MHC class I complexes on the antigen presenting cell (APC) surface for recognition by appropriate T-cells. Expression of the peptide-MHC complex on APC surface is preceded by several steps that include the generation of peptide fragments in the cytoplasm and their assembly with MHC molecules in the endoplasmic reticulum. It is now clear that MHC binding to optimally processed peptides in the endoplasmic reticulum is obligatory for their stable expression on the cell surface. However, whether a similar obligatory relationship exists between generation of processed peptides and their expression as peptide-MHC on APC surface is not known. Here, we addressed this question by analyzing the processing of ovalbumin (aa257-264, SL8) or influenza nucleoprotein (aa366-374, AM9) analogs. We examined the generation of naturally processed peptides using precursors that did, or did not, contain residues flanking the optimal MHC-binding peptides. By characterizing the peptides generated from these precursors by T-cell stimulation assays and by high performance liquid chromatography analysis, we established that intracellular assembly of peptide-MHC complexes and their expression on the cell surface can occur with peptides that lack flanking residues. The presentation of these endogenously synthesized perfect fit peptides demonstrates that the cleavage of precursor polypeptides is an independent step in the antigen presentation pathway.

To investigate the mechanism underlying neonatal T cell tolerance, we used synthetic peptides to induce tolerance. We found that induction of tolerance to one determinant on a 23-amino acid peptide did not affect the response to an adjacent determinant on the same peptide. There was no evidence of suppression of the response to the second determinant. Furthermore, even small peptides near the minimal size for a determinant, which would be very unlikely to possess a suppressor T cell-inducing determinant as well as a proliferative T cell-inducing determinant, could induce tolerance. These studies provide in vivo experiments supporting clonal inactivation as the mechanism of neonatal tolerance to immunogenic peptides.