B-cell Recognition Research Articles

Immunochemical specificity of antisera raised against the synthetic encephalitogenic peptide SH624, residues 59?74 of the myelin basic protein

Synthetic peptide SH624 (SHHPARTAHYGSLPQK), residues 59-74 of human myelin basic protein (MBP) was found to be encephalitogenic in the rabbit. Four antisera raised, against the peptide were employed in a liquid-phase equilibrium competitive radioimmunoassay with a series of synthetic peptide analogs of the region to probe the structural requirements of the B-cell determinant subsumed within SH624. The cross-reactivities of the four antisera with intact MBP were also examined. Immunochemical analyses of the four antisera suggested specificities directed against a conformational determinant dependent upon residues from the more phylogenetically conserved carboxyl C-terminal region, residues 65-74 (TAHYGSLPQK) of the synthetic immunogen. Peptide analogs shorter than SH624 from the C-terminal end showed no cross-reactivity with any of the reagent antisera while analogs shorter from the N-terminal end and including the encephalitogenic sequence TTHYGSLPQK, as well as, HYGSLPQK were reactive under equilibrium competitive conditions. SH624-reactive antibodies, cross-reactive with purified heterologous MBPs from 10 different species were also identified in all four reagent antisera. The results of these experiments support previous investigations demonstrating the accessibility of the encephalitogenic 65-74 region in intact MBP. They also underscore the importance of B-cell recognition of organ specific antigenic determinants with respect to MBP immunology and, in particular, the recognition of autoreactive determinants in the neighborhood of encephalitogenic centers.

Site recognition by protein-primed T cells shows a non-specific peptide size requirement beyond the essential residues of the site Demonstration by defining an immunodominant T site in myoglobin

In previous studies, six T sites within myoglobin (Mb) were localized. To define precisely the boundaries of the T sites, a new approach is introduced and applied here to the T site residing within residues 107-120 of Mb. Two sets of peptides were synthesized. One set represents a stepwise elongation by one-residue increments of the Mb sequence. The other set represents an identical stepwise addition of one-residue increments of the Mb sequence, but which were extended by additional unrelated (nonsense) residues to a uniform size of 14 residues. The longer peptides (nonsense-extended) usually gave higher proliferative responses than did their shorter counterparts having the same Mb region. Thus a minimum peptide size is required for optimal T-cell stimulation. The T site subtends, in three high-responder mouse strains, residues 109-119 or 110-120, depending on strain, and, in three low-responder strains, maps to residues 108-120. Thus, in this case, the T site coincides with the site of B-cell recognition and resides in a small discrete surface region of the protein chain.

The role of the epitope density and cross-reactivity between two different purine nucleosides coupled to cells

Cellular immune responses against nucleic acid antigens were analyzed in BALB/C mice. Delayed-type hypersensitivity (DTH) could be elicited by immunizing and challenging with either guanosine-coupled spleen cells (G-SC) or adenosine-coupled spleen cells (A-SC), and measured by footpad swellings. The epitope density was critical for immunization. This cellular reaction was specific to nucleosides, and cross-immunity was observed between A-SC and G-SC. In addition, cross-unresponsiveness was observed between these two nucleosides. In contrast, soluble carrier proteins coupled with either guanosine or adenosine did not induce cross-reactive immunity or unresponsiveness. The significance of the difference between these two forms of antigens in the ability to induce cross-reactivity is discussed in the context of T versus B-cell recognition in the induction or the expression of the immune response.

Antigen recognition by T cells and B cells: Recognition of cross-reactivity between native and denatured forms of globular antigens

The nature of the recognition by T cells and B cells of the native and the denatured forms of the same globular antigens, BSA or OVA, were compared. B-Cell recognition was analyzed by means of a radioimmune analysis of serum antibodies and T-cell recognition by means of an antigen-dependent proliferation assay. Rabbits hyperimmunized with native antigen in CFA produced antibodies reactive with both native and denatured antigen, with two to three times more reactivity to native antigen. Animals immunized to the denatured antigen also made antibodies reactive with both native and denatured antigens with approximately 15-fold more reactivity to denatured antigen. Inhibition and absorption studies indicated that the antibodies elicited reacted with distinct noncross-reacting antigenic determinants since unlabeled denatured antigen had no inhibitory effect on the reaction between 125I-native antigen and antibody, and vice versa. Presumably the antibodies to the alternative form of the antigen used for immunization were elicited because of small amounts of contamination with that form of the antigen present in the preparation used for immunization. T cells from BDF 1 mice primed to either native or denatured antigen demonstrated good proliferative responses to both native and denatured antigen. For example, T cells from D-OVA primed animals incorporated 51,000 cpm of [ 3H]thymidine when stimulated with D-OVA and 44,000 cpm when stimulated with N-OVA. Unlike the situation with serum antibodies, a true cross-reactivity between N and D forms of the same antigen was demonstrable at the T-cell level. This was accomplished by determining the effect of killing antigen-stimulated proliferating cells with BUdR and light on the subsequent proliferation of the same population of cells to the alternative form of the antigen. For example, T cells from N-OVA primed animals when stimulated first with D-OVA (and treated with BUdR and light) showed a marked inhibition in their subsequent capacity to respond to both antigens (93% inhibition of N-OVA response and 80% inhibition of D-OVA response). One explanation for the extensive cross-reactivity between N and D-OVA at the T-cell level is that antigen is degraded to small peptides by the proteases present in the antigen presenting macrophages. Due to their small size, no native conformational determinants are preserved so that both native and denatured antigens generate the same “sequential” determinants. However, an attempt to verify this hypothesis by inhibiting the proliferative response with anti-denatured antibodies was not successful.

IMMUNOLOGIC SIGNIFICANCE OF THE MUMPS VIRUS SKIN TEST IN INFANTS, CHILDREN AND ADULTS1

The biologic validity of cell-mediated immunity to mumps virus was evaluated in 395 children, adolescents and adults. The study protocol included the determination of cutaneous delayed hypersensitivity to viral and avian control antigens and in 79% of the subjects an essential double bleeding was performed before and after mumps virus skin test for assay of neutralizing antibody. Seven per cent of subjects expressed sufficient delayed hypersentitivity to the control antigen to erase an apparently positive mumps virus skin test. Anamnestic conversions from seronegativity to seropositivity, elicited by the mumps virus skin test, increased from 4% in children to 25% in adults, which suggests waning B-cell recognition of prior mumps virus infection in adults. Although pregnancy diminished the difference (p smaller than .001), adults showed greater cutaneous delayed hypersensitivity to mumps virus antigen than did children (p smaller than .001), suggesting that mumps virus reinfection or persistence induced the escalation of more sensitive T-cell recognition with increasing age. Humoral immunity, assessed by the double bleeding technique in the vast majority of individuals, rose form 16% (1-4 years), 45% (5-9 years) and 80% (10-14 years) to 94% in adolescents and adults. Ordinarily 75-95% in other age groups, the decline of correlation between mumps virus cellular and humoral immunity to 60% in school children may result from prior parainfluenza virus infection, inconsistent potency of the skin test antigen, concurrent immunosuppressive infection, and lagging induction of mumps virus cellular immunity in recently infected individuals. Immunologic study of a large colony of subhuman primates failed to establish an hierarchial antigenic interrelationship among mumps virus and two additional paramyxoviruses.