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A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.

T cells recognize protein antigens as short peptides processed and displayed by antigen-presenting cells. However, the mechanism of peptide selection is incompletely understood, and, consequently, the differences in the immunogenicity of protein antigens remain largely unpredictable and difficult to manipulate. In this paper we show that the susceptibility of protein antigens to lysosomal proteolysis plays an important role in determining immunogenicity in vivo. We compared the immunogenicity of proteins with the same sequence (same T cell epitopes) and structure (same B cell epitopes) but with different susceptibilities to lysosomal proteolysis. After immunizing mice with each of the proteins adsorbed onto aluminum hydroxide as adjuvant, we measured serum IgG responses as a physiological measure of the antigen's ability to be presented on major histocompatibility complex class II molecules and to prime CD4+ T cells in vivo. For two unrelated model antigens (RNase and horseradish peroxidase), we found that only the less digestible forms were immunogenic, inducing far more efficient T cell priming and antibody responses. These findings suggest that stability to lysosomal proteolysis may be an important factor in determining immunogenicity, with potential implications for vaccine design.

Sera obtained from senescence-accelerated mouse (SAM) and normal mice contained a substance that reacted with antiserum raised against ASSAM, a novel senile amyloid fibril protein isolated from the liver of SAM. This physiological substance, termed "SASSAM" (serum ASSAM-related antigenic substance), migrated to the albumin/prealbumin region in immunoelectrophoresis and the precipitation line formed with anti-ASSAM antiserum was stained positively with both Amide Black 10 B and Oil Red O/Fat Red 7B solutions, thereby suggesting that SASSAM is an alpha lipoprotein. Using Sephadex G-200 gel chromatography, SASSAM was eluted as a high mol wt form of approximately 200,000 daltons. Fractionation of lipoprotein from normal mouse serum by preparative ultra-centrifugation disclosed that SASSAM was found mainly in high density lipoprotein, HDL (the density is between 1.063 and 1.21 g/cm3). The largest amount of SASSAM was found in the HDL2 fraction (the density is between 1.063 and 1.125) and in this fraction SAA was not detected. Furthermore, ASSAM immunoreactivity appeared in the low mol wt proteins (below 10,000 daltons) of apo HDL separated in the buffer containing 8 M urea through Sephadex G-200. In 8 M urea sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), the major components of apolipoproteins in this position, possibly corresponding to apo C proteins, have the same molecular weight, 5,200 daltons, as ASSAM and this component was labeled by anti-ASSAM antiserum after transfer to nitrocellulose paper.

Two new methods are described for making chromosomal spreads of single antibody-forming cells. The first depends on the controlled rupture of cells in small microdroplets through the use of a mild detergent and application of a mechanical stress on the cell. The second is a microadaptation of the conventional Ford technique. Both methods have a success rate of over 50%, though the quality of chromosomal spreads obtained is generally not as good as with conventional methods. These techniques have been applied to an analysis of cell to cell interaction in adoptive immune responses, using the full syngeneic transfer system provided by the use of CBA and CBA/T6T6 donor-recipient combinations. When neonatally thymectomized mice were restored to adequate immune responsiveness to sheep erythrocytes by injections of either thymus cells or thoracic duct lymphocytes, it was shown that all the actual dividing antibody-forming cells were not of donor but of host origin. When lethally irradiated mice were injected with chromosomally marked but syngeneic mixtures of thymus and bone marrow cells, a rather feeble adoptive immune response ensued; all the antibody-forming cells identified were of bone marrow origin. When mixtures of bone marrow cells and thoracic duct lymphocytes were used, immune restoration was much more effective, and over three-quarters of the antibody-forming mitotic figures carried the bone marrow donor chromosomal marker. The results were deemed to be consistent with the conclusions derived in the previous paper of this series, namely that thymus contains some, but a small number only of antigen-reactive cells (ARC), bone marrow contains antibody-forming cell precursors (AFCP) but no ARC, and thoracic duct lymph contains both ARC and AFCP with a probable predominance of the former. A vigorous immune response to sheep erythrocytes probably requires a collaboration between the two cell lineages, involving proliferation first of the ARC and then of the AFCP. The results stressed that the use of large numbers of pure thoracic duct lymphocytes in adoptive transfer work could lead to good adoptive immune responses, but that such results should not be construed as evidence against cell collaboration hypotheses. Some possible further uses of single cell chromosome techniques were briefly discussed.

Details of antigen trapping and processing in the rat lymph node have been investigated by the technique of high resolution radioautography. A series of 24 adult rats was injected with 20 microg of (125)I-labeled Salmonella adelaide flagella, given as either a primary or a secondary stimulus into one hind foot-pad. At intervals ranging from 3 min to 3 wk, rats were killed and the popliteal nodes were processed for electron microscopic radioautography using Kodak NTE emulsion. The present paper deals with events in the lymph node medulla, and an accompanying report describes the radically different behavior of antigen in the cortical follicles. In the medulla, lightly labeled granulocytes were transiently encountered, but by far the greatest bulk of antigen was in macrophages. Antigen entered these cells in two ways: by direct penetration of the plasma membrane; and by pinocytosis. In either case, the antigen rapidly became surrounded by tiny vesicles which may have represented Golgi-derived "protolysosomes." Vacuolar fusion ensued and a series of progressively larger and more complex antigen-containing "phagolysosomes" was formed. Substantial amounts of antigen could be detected in such bodies for at least 3 wk. The antigen injection, as expected, caused extensive plasma-cytopoiesis. No evidence of label in plasma cells was obtained. No special anatomic relationship between plasma cells and antigen depot sites was discovered. These results are briefly discussed in relation to current theories of immune induction.

Rabbit antibodies against pneumococcus capsular polysaccharides of Types II, III, VI, VII, and XII were isolated from type-specific antisera by precipitation with homologous antigen and hydrolyzed with either subtilisin, chymotrypsin or trypsin. Purified antibodies were also hydrolyzed with papain; the two polypeptides with active antibody sites and the crystallizable polypeptide were separated, studied in the ultracentrifuge and hydrolyzed with either subtilisin or chymotrypsin. The resulting peptides were separated on filter paper by electrophoresis and chromatography. 1. The two polypeptides with antigen-combining activity, fractions I and II, each constituted about 25 per cent of the original antibody molecule; the crystallizable polypeptide did not combine with antigen, fraction III, and represented about 35 per cent of the original antibody molecule. About 10 to 15 per cent of the original antibody molecule was hydrolyzed by papain into about 35 peptides. 2. The peptide patterns obtained for hydrolyzates of fraction I were almost identical with those obtained for fraction II obtained from the same antibody and were quite different for those obtained for fraction III. 3. Many of the peptide spots in the patterns obtained with whole antibody hydrolysates contained at least two peptides derived from different parts of the antibody molecule. 4. Differences were observed in the peptide patterns for different antibodies that suggested the existence of differences in both primary and tertiary structures among these antibodies.

The Ehrlich ascites tumor, which has been subjected to prolonged selection for growth in allogeneic hosts, possesses powerful mechanisms for the suppression of antigens normally found on the cell surface. It has previously been shown (1, 2) that when cells in which surface antigens are fully expressed are fused with Ehrlich cells, the suppressive mechanisms of the latter continue to operate and the new surface antigens introduced into the hybrid cell by the other partner are also suppressed. In the present paper we describe the properties of hybrids in which one parent cell was the TA3 ascites carcinoma. There are two sublines of this carcinoma which originally arose as a spontaneous mammary carcinoma in a strain A mouse. The TA3/St line has a high concentration of H-2(a) antigens and shows a strain-specific transplantation behavior; the TA3/Ha subline has a drastically reduced antigen concentration and readily transgresses histoincompatibility barriers. The immunoresistant TA3/Ha subline arose spontaneously without having been subjected to any known immunological selection pressure. Hybridization of TA3/Ha cells with normal diploid ACA fibroblasts reestablished full expression of H-2(a) antigens in nine independently derived hybrid clones. Full reestablishment of both D- and K-end components of the H-2(a) complex could be demonstrated. In some hybrid clones the concentration of H-2(a) antigens was found to be comparable to that seen in (A x ACA)F(1) fibroblasts, whereas in others a higher concentration was observed, even exceeding, in some cases, the levels found in the TA3/St line. The H-2(f) complex, contributed by the ACA parent cell, was fully expressed in eight of the nine hybrid clones studied. Antigen suppression thus behaves as a recessive character in the TA3/Ha hybrids, whereas in the Ehrlich hybrids antigen suppression is dominant.

The present paper deals with reactions between antiserums and the capsules of Torula histolytica (Cryptococcus hominis or Torulopsis neoformans) and of Sporotrichum schenckii. The reactions, although similar in principle to the capsular swelling seen in Quellung tests of bacteria, present a special interest because the microorganisms are larger in size and more complex in structure than bacteria. That the reactions on the capsules were caused by immunologically specific antibodies was shown by the fact that the capsular reactivity of the serums was directly related to their capacities to agglutinate suspensions and to precipitate soluble antigens of Torula and of Sporotrichum, and by the fact that the capsular reactivity was removed from the serums by absorption with soluble antigens of the respective fungi. The soluble antigens included partially purified products from broth cultures of Torula and of Sporotrichum, a partially purified product from Sporotrichum-infected mice, and a purified polysaccharide from a culture of Torula grown in a synthetic medium. The purified polysaccharide was highly reactive in precipitation tests with unabsorbed serum, and its ability to absorb the Torula capsular reactivity from the serums indicates that the major antigens on the surface of encapsulated Torula cells are polysaccharides, identical with or similar to the product used in the present study.

The findings presented in this paper indicate that 60 gm. of liver tissue, taken from adult donor rabbits 2 weeks after injecting them with large amounts of bovine gamma-globulin, and transferred to the peritoneal cavities of recipient rabbits prepared for a secondary reaction, contain enough of the antigen to induce the formation of detectable amounts of precipitin in the recipient rabbits. On the other hand, liver taken from donors 21 and 28 days after injecting them with the same antigen fails to bring about a similar effect. The findings outlined in the paper suggest that the antigen is degraded after the 2nd week to such an extent that it fails to engender the formation of complete antibody in the recipient animals, although previous work has shown (2-4) that the transfer of liver tissue from similar donor rabbits to mice renders the recipient mice anaphylactically sensitive to the antigen, bovine gamma-globulin. The implications of the findings are discussed.

The present experiments have shown that phagocytosis occurs in the absence of specific antibody and in the absence of a "suitable physical surface", as further that the presence of a rough surface does not increase the in vitro phagocytosis of pneumococci by polymorphonuclear leukocytes. This held true during repetition of Wood's experiments, as well as when more controlled quantitative techniques were employed, when conditions were made optimal for phagocytosis by increasing bacterial concentrations, and when blood leukocytes were substituted for exudate leukocytes. Evidence has been presented previously that the stimulation of phagocytosis of E. coli, B. abortus, and Type IV Pneumococcus, after contact with filter paper or an active compound present in filter paper, is a chemical effect rather than a physical effect. This type of stimulation did not occur with the Type I A5 Pneumococcus. The leukocyte of the circulating blood was found to be definitely superior to the exudate leukocyte in phagocytic capacity, under all the experimental conditions tested.

Starch electrophoresis was used to separate allergic sera into electrophoretically distinct albumin, alpha-, beta-, and gamma-globulin fractions. The identity of the various fractions was established by free and paper electrophoresis. The gamma-globulin fractions were also characterized by ultracentrifugation. The serum skin-sensitizing antibody to ragweed pollen was shown to be associated with the gamma-globulins in fourteen allergic sera by means of passive transfer technique. The mobility of the skin-sensitizing antibody corresponded to that of gamma-1 and/or gamma-2 globulins. All the other serum fractions were devoid of reaginic activity. Reaginic activity per se was not directly related to protein concentration. The discrepancies between these results, and those obtained by others and ourselves previously are discussed.

High and low zone antibody tolerance to bacterial flagellin can be induced in adult strain W Wistar rats by multiple injections of a cyanogen bromide (CNBr) digest of flagellin at two widely spaced dose levels. Intermediate doses of the CNBr digest produce enhanced antibody titers to flagellin rather than antibody tolerance. Studies reported in this paper revealed that both high and low zone antibody tolerance to flagellin were accompanied by heightened levels of delayed-type hypersensitivity. Conversely, when enhancement of the antibody response occurred, suppression of delayed hypersensitivity was observed. This inverse relationship between humoral and cell-mediated immunity was very striking in strain W Wistar rats but was not quite so clear-cut in another strain of Wistar rats (strain J). Strain J rats were resistant to the induction of antibody tolerance and gave higher immunological responses to flagellin than strain W animals. In addition, it was observed that, in contrast to adult tolerance, administration of the CNBr digest to neonatal rats induced complete tolerance at the level of both humoral and cell-mediated immunity. These findings were discussed in the light of earlier studies with flagellin and provide further evidence for a previously described hypothesis.

This paper describes the trapping of antigen in lymphoid follicles of rat popliteal lymph nodes as revealed by electron microscopic radioautographs following injection of (125)I-labeled Salmonella adelaide flagella and other materials. The antigen was taken up vigorously, and to an approximately equal extent, by both primary and secondary follicles. The rate of uptake was faster in preimmunized than in virgin adult rats. The bulk of the antigen in follicles was extracellular, and persisted in this location for at least 3 wk. Label was most frequently found at or near the surface of fine cell processes. Many of these were branches of dendritic follicular reticular cells. Such processes interdigitated with equally fine processes of lymphocytes, creating an elaborate meshwork. In some cases, antigen was found between lymphocytes which appeared to be in close apposition. Occasionally, a few grains appeared over lymphocyte nuclei and study of serial sections suggested that this probably represented true entry of small amounts of antigen into lymphocytes. The characteristic "tingible body" macrophages (TBM) of germinal centers appeared to play only a secondary role in follicular antigen retention. They showed degrees of labeling over their phagocytic inclusions varying from negligible to moderately heavy. Moreover, follicles lacking or poor in TBM retained antigen just as effectively as those containing numerous TBM. The hypothesis is advanced that TBM may be derived from monocytes that migrate down from the circular sinus. Follicular localization of three other materials was also studied, though not in such detail. These were (125)I-HSA complexed to anti-HSA: (125)I-labeled autologous IgG; and (125)I-monomeric flagellin. All of these showed the basic features of intercellular, membrane-associated deposition noted with (125)I-flagella. The role of follicular antigen depots in immune induction is discussed. The tentative conclusion is reached that follicular antigen in a primary follicle encounters natural antibody on the surface of certain antigen-reactive lymphocytes. The resultant reaction causes blast cell transformation and eventually the genesis of a germinal center.

Experiments in rabbits were designed to test the two unproven assumptions of the hypothesis proposed in the companion paper (1): that Original Antigenic Sin is fundamentally a restricted anamnestic response, and that there exists a trapping mechanism capable of deflecting antigen from one kind of cell and guiding it to another. It is shown that whole-body X-irradiation, sufficient to abolish primary but not secondary production of antibodies, leaves all manifestations of the Original Antigenic Sin untouched. This proves the first assumption. Primary immune animals challenged with very large doses of a related antigen produce an immediate response of cross-reactive antibodies, followed by a standard primary response to the challenging antigen. When boosted with an appropriate mixture of both antigens, the response is of standard secondary type to the homologous antigen, followed by a standard primary response to the crossreacting antigen. When animals are primarily vaccinated with a mixture of two related antigens, small booster doses of either will stimulate a standard secondary response only. When such animals are given very large booster doses of either antigen, the response is a compound of a homologous secondary and of an Original Antigenic Sin-type against the related antigen. Each of these findings demonstrates a corollary of the second assumption. The results are discussed in terms of the limitations they impose on theories concerned with the production of antibodies.

The DNA-synthesizing capacity of single antibody-forming cells was tested by a combination of micromanipulatory and autoradiographic techniques. Rats were immunized with S. adelaide flagellin, a protein antigen known to contain significant contamination with somatic (O) antigen. Single cells from secondarily immunized rats were tested for production of anti-H and anti-O antibodies by previously described and newer techniques. Positive antibody producers were transferred onto clean dry slides by micromanipulation, and autoradiographs were performed. When rats had received tritiated thymidine 1 hour before killing, labeling of antibody-forming cells was taken to imply that the cell was preparing for further mitotic division. It was found that on the 2nd and 3rd day of a secondary response, many of the antibody-producing cells in the nodes (chiefly plasmablasts) were incorporating tritiated thymidine. At the height of the cellular response, however, at 4 and 5 days, the majority of active antibody producers (chiefly mature plasma cells) were incapable of DNA synthesis. There appeared to be an inverse relationship between the antibody-forming and DNA-synthesizing capacities of the cell population under study; as more of the cells studied formed detectable antibody, fewer of them incorporated the DNA precursor. The age of plasma cells was also studied. Animals were killed at the height of the cellular immune response, having previously received an injection of tritiated thymidine 1 to 48 hours before killing; i.e., at 63 to 110 hours after their secondary stimulus. As the interval between isotope injection and killing increased, the proportion of antibody-forming cells showing labeling increased. With an interval of 30 hours, about half the antibody-forming cells were labeled and of 48 hours, over 95 per cent were labeled. This was taken as evidence that, few, if any, antibody-forming cells found at the height of a secondary response were more than 48 hours old. On the basis of these experiments and those reported in the accompanying paper, a simplified scheme showing the development of an antibody-forming clone in the secondary response was proposed.

Previous studies suggested that an active immune response was partially responsible for maintaining immunological unresponsiveness to sheep erythrocytes. Measurement of the plaque-forming (antibody-releasing) cell response proved to be a sensitive indicator of an immune response to sheep erythrocytes in the absence of detectable circulating antibody to the antigen. The present studies were undertaken to determine whether an active immune process, measured by the plaque-forming cell response, was partially responsible for induction and maintenance of tolerance. Rats injected intraperitoneally with large doses of sheep erythrocytes beginning at the day of birth develop tolerance to the antigen. In this paper, the plaque-forming cell and antibody response to sheep erythrocytes was characterized for rats receiving a single antigen injection at various ages and for rats which received repeated antigen injections as adults. The dose of antigen was the same as that used to produce tolerance; the injection schedule for repeated immunizations was also the same as that used to produce tolerance. Rats receiving a single antigen injection on the day of birth or at age 7 days had no measurable response to the antigen. Rats receiving a single antigen injection at age 17 days and sacrificed 4 days later had an unequivocal response to the antigen. The spleens had about one-tenth as many plaque-forming cells as spleens of adult animals immunized similarly, but the antibody titers were as high as titers for adult animals. Presumably the high titers of these young animals resulted from the high ratio of plaque-forming cells to body weight and blood volume. Adult animals receiving a single antigen injection had a peak or near peak plaque-forming cell response 4 days after immunization; at this time, sera contained high titers of 19S antibody and the numbers of plaque-forming cells in spleens correlated reasonably well with circulating antibody titers. 7S antibody appeared in serum 5 or 6 days after immunization. The numbers of plaque-forming cells declined progressively 2 and 3 weeks after immunization. Repeated twice weekly, injections of the antigen in adult rats produced a marked decline and then stabilization of numbers of plaque-forming cells in spleens. Although the numbers of plaque-forming cells were fewer, titers of 19S and 7S antibody stabilized at high levels. A progressive recovery of the plaque-forming cell response and a rise in antibody titer occurred when the interval between the last 2 injections was increased from 3 to 10, 17, or 32 days. These findings suggested that repeated closely spaced antigen injections interfered with either cell division or maturation of antibody-forming cells. As the interval between injections was increased, additional antibody-forming cells matured or were formed through cell division. Thus, relatively constant antigenic stimulation provided a mechanism for controlling or limiting the response of antibody-forming cells. In the following paper, it will be shown that this mechanism operating together with a homeostatic mechanism which prevents induction of new antibody-forming cells serves to regulate the immune response to repeated injections of antigen.

Quantitative relationships between the amounts of encephalitogenic neural tissue and mycobacterial adjuvant are presented for the guinea pig, for comparison with similar results obtained in the mouse and presented in an accompanying paper by Lee and Schneider (27). Definite threshold amounts of both neural encephalitogenic and mycobacterial adjuvant can be defined. With the proper amounts of each component, 100 per cent of guinea pigs can be made to develop EAE, 97 per cent dying of it, and over 50 per cent becoming paralyzed by the 12th day after challenge. With moderate amounts of encephalitogen the severity and incidence of EAE can be very great, but this encephalitogenic potential can be masked if large amounts of mycobacteria are employed. The mechanism of this masking effect by excess adjuvant is not known, but speculation centers upon the possibility of competition of antigens of the mycobacteria at the expense of those of the encephalitogen.

As evidenced by precipitin analysis with pooled human gamma globulin, at least 12 distinct antigens were produced in cultures by one strain of Group A streptococcus (C203S). It was suggested on this basis, that these antigens were produced in vivo during human infections. By the combined use of continuous flow electrophoresis on paper curtains, and column chromatography with calcium phosphate gels, five of these have been isolated in a probable high state of purity. One of the components was obtained from culture filtrates of a Group C streptococcal strain. Three of the purified antigens have been tentatively identified as streptolysin "O", diphosphopyridinenucleotidase, and proteinase precursor. The latter could be very readily crystallized, and appears "identical" with that described by Elliott. The DPNase was of extremely high potency, 1 mg. being capable of destroying 12.6 gm. of DPN in 7(1/2) minutes at 37 degrees C. The identity of the other two components is uncertain as yet. They are distinct from each other and the above products immunologically, and are not related to the "C" carbohydrate. The applicability of these methods for the analysis of infectious diseases generally was discussed.