Altered Antigen Research Articles

Protection against transplanted and spontaneous lymphoma by inoculation of heat-altered syngeneic tumor cells in splenectomized mice.

AKR mice with a high incidence of spontaneous lymphoma and leukemia were treated with various combinations of heat-altered syngeneic tumor cells, BCG, and splenectomy. It was found that the combination of heat-altered tumor cells and splenectomy markedly inhibited death secondary to both transplanted and spontaneous disease. BCG in combination with altered tumor cells was moderately effective in suppressing transplanted tumors but had no protective effect against spontaneous tumors. Neither splenectomy nor BCG alone offered any protection against spontaneous disease. The results are especially interesting in view of the evidence that there may be immunological tolerance to tumor specific antigens on these leukemia cells. The results may represent the induction of immune materials against an altered tumor antigen which cross-reacts with unaltered tumor antigens on viable leukemic cells and aids in their destruction. Splenectomy may exert its beneficial effect by reducing circulating enhancing antibodies.

Altered antigen binding by immunocompetent cells as a reflexion of immunological history.

AT least one of the cell types involved in the immune response has antigen specific receptors. This can be concluded from experiments in which specific immune responses have been dramatically affected by manipulations dependent on the ability of such cells to selectively bind to antigen. These manipulations include elimination of cells bearing a receptor of one specificity by passage through a column in which the homologous antigen is bound to particles1; selective lethal irradiation of a sub-population of cells consequent to their ability to bind radioactively labelled antigen2; and separation of cells capable of forming rosettes by binding to foreign erythrocytes3–4. We have used this latter technique to investigate whether the nature of the binding sites on cells necessary for the response to sheep erythrocytes in mice is influenced by the immunological history of the animal.

Serological and finger-printing analyses of mutant flagellar antigens of Salmonella.

SUMMARY: Spontaneous mutants with altered flagellar antigen were isolated from a phase-1 stable Salmonella strain, tr 6, whose phase-1 antigen consists of at least three antigenic factors, g3, g4 and f. Three kinds of antisera reacting with each of these factors were used as the selective agents. The mutants were isolated by picking from swarms in semisolid medium containing the respective antisera. Mutants were classified into two groups. A group comprising four mutants selected with antiserum to g3 and two mutants selected with antiserum to g4, in which all three antigenic factors examined were simultaneously altered; and a second group comprising all the other mutants, in which only a single factor corresponding to the selective antiserum was altered. All the altered factors retained weak cross-reactivity with the original factors. In all cross-absorption-agglutination tests between each of the mutant antigens and the wild-type antigen, activity of the antisera was fully absorbed, showing that the mutant antigens had neither lost any antigenic specificities nor gained any new specificity. Transductional analysis showed that the sites of mutation were inseparable from H 1, the structural gene for phase-1 flagellin. Tryptic peptide patterns of flagellins of two mutants were compared with that of the wild-type strain and a difference of one or two peptides was detected. From these results it is concluded that these mutants possessed a minor change in primary structure of flagellin which resulted from a mutation in the H 1 gene.

Cellular compartmentalization of herpesvirus antigens during viral replication.

HEp-2 cells infected with herpes simplex virus develop five distinct immunofluorescent elements. Three (small nuclear granules, large nuclear granules, and an amorphous mass filling the nucleus) contain antigens which react with a rabbit serum prepared against boiled infected cell debris. A labeled pool of human antibody revealed antigens making up cytoplasmic granules and those responsible for a diffuse cytoplasmic fluorescence. All five immunofluorescent elements are demonstrable with a rabbit serum prepared against unheated infected cell debris. Viral antigens are segregated in the nucleus or in the cytoplasm; within the limits of detection, each antigen accumulates in one compartment only. The antigens responsible for the diffuse cytoplasmic fluorescence and for the amorphous nuclear mass are synthesized early in infection; they are formed in arginine-deprived cells and exist in a form which does not sediment on centrifugation at 79,000 x g for 2 hr. The antigens comprising the nuclear and cytoplasmic granules arise relatively late in infection; they are not formed in arginine-deprived cells, and they are readily sedimented on centrifugation at 79,000 x g for 2 hr. Heating (60 C for 2 hr) confers on one or more cytoplasmic viral antigens a new specificity; the altered antigens are demonstrable with labeled rabbit anti-boiled infected cell serum which normally does not combine with cytoplasmic antigens.

Immune response to polypeptidyl proteins in rabbit tolerant to the protein carriers

Abstract : A study was made of the immune responses to polypeptidyl proteins in rabbits which were either naturally tolerant or made experimentally unresponsive to the protein carriers. Experimentally acquired tolerance to HSA could be terminated by immunization of the unresponsive rabbits with poly-Ltyrosyl derivatives of HSA. Two parameters determined the breakdown of tolerance: (1) the chemical nature of the peptides attached: polytyrosyl HSA was effective in the termination of tolerance to HSA, whereas polyalanyl HSA was not; (2) the degree of enrichment was tyrosine. There appears to be an optimal degree of enrichment, i.e. of molecular alteration of the HSA, which will confer on the altered antigen the maximal potency to terminate tolerance: a slight alteration (3 per cent enrichment of HSA with Tyr) and a severe alteration (13 per cent enrichment) were less effective in tolerance breakdown than an intermediate degree of alteration (7 per cent enrichment with Tyr). The level of tolerance breakdown obtained by polytyrosyl HSA, as measured by the ratio of anti-HSA/ anti-polytyrosyl HSA, was greater than the level obtained previously by other chemically altered antigens. The significance of this ratio was discussed in relation to the cellular basis of immunological tolerance. Antibodies were produced to the peptides per se, when attached to proteins towards which the animal is naturally tolerant (RSA), or to proteins to which the animal has acquired tolerance (HSA). In both systems, there was a similar pattern of antibody formation, which may reflect a similarity in mechanism between natural and actively acquired tolerance.

Termination of acquired immunological tolerance to protein antigens following immunization with altered protein antigens.

Acquired tolerance to BSA in rabbits was terminated following the injection of certain preparations of altered BSA. Injections of Freund's adjuvant containing BSA complexed to anti-BSA, heat-denatured BSA, acetyl-BSA, picryl-BSA, or arsanil-BSA failed to terminate the tolerant state. Except in an occasional tolerant rabbit, injections of these preparations failed to cause the production of precipitating antibody to the altered preparation. Similar results were obtained following injections of alum-precipitated preparations of pepsin-degraded BSA, acetyl-BSA, and picryl-BSA. Injections of Freund's adjuvant containing sulfanil-BSA terminated the tolerant state, but only small amounts of non-precipitating anti-BSA were produced. Injections of Freund's adjuvant containing picryl-acetyl-BSA terminated the tolerant state in two of six rabbits, but again, only small amounts of non-precipitating anti-BSA were produced. Injections of an alum-precipitated preparation of picryl-acetyl-BSA failed to terminate the tolerant state. On the other hand, injections of Freund's adjuvant containing arsanil-sulfanil-BSA terminated the tolerant state in eleven of eleven rabbits and caused the production of precipitating anti-BSA in all nine of the rabbits tested. The tolerant state was terminated also in six of six rabbits injected with an alum-precipitated preparation of arsanil-sulfanil-BSA. Only one of these rabbits produced precipitating anti-BSA. In addition, the injection of BGG-tolerant rabbits with arsanil-BGG, sulfanil-BGG, or arsanil-sulfanil-BGG terminated the tolerant state. These results were discussed in relation to both the clonal selection theory of antibody production and autoimmunity.

Immunochemical Studies of Poliovirus

Summary Exposure of purified poliovirus, Fraction SDGD, which comprises ID+ and NID+ particles, to heat at 56°C or to ultraviolet light, results in loss of infectivity and in a change in immunologic specificity, manifested by a loss of reactivity in complement fixation tests with suitable convalescent phase sera from poliomyelitis patients, and a concomitant appearance of reactivity with certain acute patient phase sera. The rate of loss of infectivity on heating at 56°C was about the same as the rate of conversion of immunologic specificity. On the other hand, upon exposure to ultraviolet light the rate of loss of infectivity was far greater than the rate of serologic change. The altered antigen has been designated as denatured D-antigen. We consider it likely that the NIC- particles described in the previous report (9) are immunologically identical with, or similar to, denatured D-antigen.

Reversibility of the Alpha and Beta Phases of Salmonella typhi.

By the cultivation of Salmonella typhi in broth containing antiserum derived from the specific phase of Salmonella muenchen, Kauffmann was able to isolate a variant which possessed altered flagellar antigens and which was no longer agglutinable in S. muenchen antiserum. The variant was obtained from only one culture and was not reversible. It was designated as a beta phase of the theretofore supposedly monophasic typhoid bacillus. Through cultivation in immune serums, beta phases were also obtained by Kauffmann and Tesdal from Salmonella schleissheim and by Gard from Salmonella abortus-canis. None of these induced phases were reverted to the naturally occurring phases of the bacilli and it is questionable whether they represented phase-variation as it is observed in naturally diphasic species, or whether they were mutants produced by exposure to antiserums. It was found by Bruner and Edwards that the technic of Wassén was very effective in the isolation of suppressed specific phases in the so-called monophasic nonspecific Salmonella types. Therefore 7 cultures of S. typhi in our possession were examined by this method. The organisms were cultivated in agglutination-tubes containing semi-solid agar to which had been added sufficient S. muenchen antiserum to confine the growth of the normal, or alpha, phase of the bacilli to the line of inoculation. The medium was inoculated by stabbing at one side of the tube. Outgrowths from this line represented variation in the flagellar antigens of the bacilli. By this method beta phases similar to the one described by Kauffmann were obtained from all the cultures on the first trial. Subsequently, the experiment was twice repeated with the same results. The variants were transferred several times in semi-solid agar containing S. muenchen antiserum to free them of the alpha phase.