A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY : I. Specificity and Sensitivity

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (⁶¹Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man.     

SEROLOGIC EVIDENCE FOR ANTIGENS CONTROLLED BY THE Ir REGION IN MICE

Antibodies produced in B10.D2 mice against soluble lymphocyte membrane antigens of B10.A (H-2^a) mice reacted only with lymphocytes of the strains carrying the Ir^k region, i.e., B10.A(2R), B10.K, B10.BR, B10.HTT, AQR, A.TE, C3H, and CBA; they did not react with cells of strains carrying different Ir regions, i.e., B10.A(4R), B10, B10.M, A.SW, DBA/1. It is therefore concluded that the antigen detected with these antibodies is apparently controlled by the Ir region of the H-2 complex. The antigen is present on some T lymphocytes and absent on B lymphocytes. Its presence or absence seems to correlate with MLC and GVH reactivity.     

A STUDY OF THE ANTIGENS INVOLVED IN ADENOVIRUS 12 TUMORIGENESIS BY IMMUNODIFFUSION TECHNIQUES

The use of the agar gel diffusion technique has established the presence of three distinct antigenic reactions in the sera of Ad. 12 tumor-bearing hamsters. Only one of these antigens is directly demonstrable in the tumor. This "tumor" antigen is also formed during early stages of the infectious cycle in tissue culture cells. Other antigens present in the tumor, but only demonstrable indirectly with the use of antibody-containing serum of tumored hamsters, are the classical type-specific C antigen, and a new antigen, termed D. Of ninety-eight Ad. 12 tumored hamster sera, six reacted in gel diffusion with virus and tumor preparations, and thirty-one with tumor only. Sera which reacted in gel diffusion with viral antigen uniformly bad neutralizing antibody and high titers of CF antibody against viral and tumor antigens; however, many sera with comparable antibody titers did not react with the virus in gel diffusion. Sera which reacted in gel diffusion only with tumor antigen also had high CF antibody titers, but there was no correlation with neutralizing antibody.     

RELATION OF CHROMOSOME 4 (LINKAGE GROUP VIII) TO MURINE LEUKEMIA VIRUS-ASSOCIATED ANTIGENS OF AKR MICE

Genes specifying or controlling the expression of G_IX (cell surface), GCSA (cell surface), and gs (internal viral) antigens are located in chromosome 4 (linkage group [LG] VIII) of the AKR mouse. All three antigens may exhibit mendelian inheritance, mice being antigen positive or antigen negative, but each may also appear in leukemic cells of mice whose inherited genotype was antigen negative. The G_IX-determining gene in LG VIII of AKR mice apparently is equivalent to Gv-1, which determines expression of the same antigen in 129 strain mice, but which in the latter strain is located in LG IX. As the estimated distance of Gv-1 from H-2 in 129 mice is considerable (37 units) further tests are now indicated to assess the possibility of pseudolinkage in this case. The Fv-1 locus, also located in LG VIII, influences the mouse's titer of MuLV, and might thereby be thought to regulate the G_IX and gs phenotypes of AKR backcross segregants. But the data indicate a discrete LG VIII locus for G_IX, since expression of this antigen is mendelian and independent of infectious virus titer. Since the G_IX and GCSA phenotypes of AKR backcross segregants were invariably concordant, these two antigens must be specified or controlled by closely linked genes, and the latter also is presumably independent of virus titer. The question as to what extent expression of gs antigen in the segregants is secondary to virus production is undecided.     

TWO KINDS OF ANTIGEN SUPPRESSION IN TUMOR CELLS REVEALED BY CELL FUSION

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)F₁ 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.     

ISOANTIGENS OF THE H-2 AND Tla LOCI OF THE MOUSE : INTERACTIONS AFFECTING THEIR REPRESENTATION ON THYMOCYTES

H-2 and TL isoantigens of the mouse are specified by the closely linked genetic loci H-2 and Tla. A. study of their representation on thymocytes was performed in order to reveal any interactions between the determinant genes or their products affecting the synthesis or disposition of these components of the thymocyte surface. The method employed was quantitative absorption of cytotoxic antibody by viable thymocytes. The phenotypic expression of TL antigens was found to reduce the demonstrable amount of certain H-2 antigens to as little as 34% of the quantity demonstrable on TL- thymocytes. A reduction was observed in all three H-2 types tested, (H-2^b, H-2^a, and H-2^k). As antigenic modulation (change of TL phenotype from TL+ to TL-, produced by TL antibody) is known to entail a compensatory increase in H-2(D) antigen, it is concluded that the TL phenotype, rather than the Tla genotype, influences the surface representation of H-2 antigens. The two known TL+ phenotypes of thymocytes (TL.2 and TL.1,2,3) depress H-2 equally. The H-2 specificities affected are those determined by the D end of the E-2 locus, which is adjacent to Tla; antigens of the K end, which is distal to Tla, are not depressed. The reduction of demonstrable H-2 antigen on the thymocytes of TL+ x TL- progeny is half that of thymocytes of TL+ x TL+ progeny and the reduction affects equally the products of both H-2 alleles (cis and trans in relation to Tla), indicating that the mechanism of H-2 reduction by TL is extrachromosomal. Whether it involves diminished synthesis of H-2 or steric masking by TL at the cell membrane is unknown, but in either case the reciprocal relation of TL and H-2(D) antigens implies that they probably occupy adjacent positions on thymocytes and that the gene order, H-2(K): H-2(D):Tla is reflected in cell surface structure. Extrachromosomal interaction, apparently involving control of synthesis, occurs also within the TL system of antigens. Thymocytes of TL.2 x TL.1,2,3 progeny express the full homozygous quantity of antigens TL.1 and TL.3 (but not of TL.2), in contrast to the half-quantity present in thymocytes of TL- x TL.1,2,3 progeny. Another example of interaction is implicit in the finding that thymocytes of TL-1,2,3 x TL.1,2,3 progeny have more TL.2 antigen than thymocytes of TL.2 x TL.2 progeny, but in this instance there is nothing to indicate whether the mechanism is chromosomal or extrachromosomal. Thus the quantitative surface representation of at least some H-2 and TL antigens is influenced by the cellular complement of H-2:Tla genes as a whole. Comparison of H-2 heterozygous thymocytes with H-2 homozygous thymocytes in quantitative absorption tests shows (a) more than the expected 50% of each parental-type H-2 antigen on heterozygous cells, and (b) a greater suppression of H-2 by TL in H-2 heterozygotes in comparison with H-2 homozygotes. Both results may be explained on the basis of differences in the density of H-2 antigenic sites and consequent differences in the efficiency of absorption of H-2 antibody. These considerations may be useful in other contexts, e.g. in estimating the representation of Rh antigens on the red cells of human subjects homozygous and heterozygous for Rh components.     

DIFFERENTIAL EFFECTS OF INHIBITORS ON THE STEPS LEADING TO THE FORMATION OF SV40 TUMOR AND VIRUS ANTIGENS

The effect of DNA antagonists and various antibiotics on steps in the synthesis of SV40 virus in green monkey kidney cells was investigated. Both the early forming tumor (T) antigen, as well as the later synthesized virus (V) antigen, were synthesized in the presence of fluorouracil and iododeoxyuridine. Cytosine arabinoside (and fluorodeoxyuridine in starved cells) prevented synthesis of V antigen but not T antigen. The synthesis of T antigen therefore does not require synthesis of virus DNA. Virus particles formed only in the presence of the iododeoxyuridine and they were non-infectious. Actinomycin D inhibited synthesis of both tumor and virus antigens, suggesting that the synthesis of these antigens involves DNA-dependent RNA. Puromycin allowed synthesis of the T antigen which remained localized at the nucleolar membrane. This finding with puromycin suggests that the T antigen is a protein of low molecular weight. Virus antigen forming in the presence of mitomycin C, p-fluorophenylalanine, iododeoxyuridine, or fluorouracil was distributed atypically. These inhibitors caused the V antigen to be diffusely spread throughout the nucleus, or to be concentrated at the nuclear membrane.     

TRANSFORMATION OF A HISTOCOMPATIBILITY IMMUNOGEN INTO A TOLEROGEN

H-2 antigens on spleen cell membranes absorb antibody to H-2 antigens and induce both humoral and cellular responses. Liver cell membrane H-2 antigens by contrast also absorb antibody but do not influence cellular response and are tolerogenic for the humoral response. This paper demonstrates that syngeneic liver cells contain a substance which can transform the properties of allogeneic spleen cell membranes into those of allogeneic liver cell membranes, i.e., transform a humoral immunogen into a humoral tolerogen. The process appears to be accompanied by cleavage of an antigen component from the spleen membrane and hence to result in a structural change in the H-2 antigen.     

ANTIGENIC MODULATION : LOSS OF TL ANTIGEN FROM CELLS EXPOSED TO TL ANTIBODY. STUDY OF THE PHENOMENON IN VITRO

Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions.     

EVIDENCE FOR IDENTITY OR CLOSE ASSOCIATION OF THE Fc RECEPTOR OF B LYMPHOCYTES AND ALLOANTIGENS DETERMINED BY THE Ir REGION OF THE H-2 COMPLEX

Immunoglobulin complexes, composed of heat-aggregated human Ig, were shown to bind to mouse B lymphocytes of a variety of strains, but not to either thymocytes or thymus-derived (T) lymphocytes under a variety of conditions. It was shown that this binding was not due to either natural human antibodies against mouse nor to nonspecific binding of human Ig by mouse lymphocytes. Such complexes were shown to bind to the same sites which bind mouse antibody-antigen complexes. This site is known as the Fc receptor. The binding of Ig complexes to mouse B lymphocytes was markedly inhibited by pretreatment of the lymphocytes with anti-H-2 antisera. A series of experiments indicated the specificity of this result, including the fact that this inhibition was shown not to be due to the artifact of shedding of H-2 antibody-antigen complexes, nor to nonspecific steric inhibition. The antibodies within anti-H-2 antisera which were responsible for this inhibition were specific for alloantigens associated with the Ir region of the H-2 complex (Ia antigens). Antiserum specific for these Ia antigens produced inhibition, whereas antisera specific for antigens determined by the K or D regions of the H-2 complex did not. Evidence was obtained using F₁ hybrid cells that at least some Ia antigens of both parental types are expressed on every B lymphocyte (i.e. codominant expression). These data indicate that the Fc receptor and a series of alloantigens controlled by the Ir region of the H-2 complex are identical or closely associated on the B-lymphocyte surface membrane. This observation may have implications for the mechanism of control of the immune response.     

VIRAL AND CELLULAR SURFACE ANTIGENS OF MURINE LEUKEMIAS AND MYELOMAS : SEROLOGICAL ANALYSIS BY IMMUNOELECTRON MICROSCOPY

Immunoelectron microscopy (IEM) of mouse cells which were productively infected with murine leukemia virus (MuLV) yielded the following conclusions: See PDF for Structure With two exceptions, the alloantigens H-2, θ, Ly-A, and Ly-B were not present on complete or incomplete virions produced by cells bearing these antigens.The exceptions were H-2^k (but not H-2^b) and θ both appeared on only a minority of virions and never on more than a small part of the circumference. The incidental discovery of an additional envelope antigen on virions produced by a BALB/c mouse myeloma but lacking from passage A Gross virions distinguishes these two viruses as MuLV subtypes; it also illustrates that IEM can be applied as a primary tool for antigenic analysis, as well as for its usual purpose of finding out where antigens are situated.     

INDEPENDENCE OF H-2K AND H-2D ANTIGENIC DETERMINANTS ON THE SURFACE OF MOUSE LYMPHOCYTES

At 37°C, fluorescein-conjugated anti-H-2 alloantibodies specifically induce, at the surface of living mouse lymphocytes, the redistribution of the corresponding H-2 antigens, which cluster as patches and sometimes single caps at one pole of the cell. This aggregation is inhibited at 0°C and the H-2 antigens, stained by fluorescent antibodies in the cold, appear evenly spread over the cell surface. This phenomenon was used to define the relationships between the membrane structures bearing the antigens coded by the H-2K and the H-2D genes of the H-2 region. Monospecific anti-H-2 antibodies coupled to either tetramethyl rhodamine isothiocyanate or fluorescein isothiocyanate were used to induce the redistribution of H-2D and H-2K antigens of the H-2^b and H-2^k haplotype at the surface of lymph node cells from homozygous and F₁ hybrid mice. It was observed that the diffuse distribution of H-2K antigens labeled at 0°C was not affected by the prior antibody-induced aggregation of H-2D antigens and vice versa. The results were the same for H-2 antigens governed by genes located either in cis or in trans position. These data indicate that the H-2K and H-2D antigens migrate independently at the cell surface, and suggest that the gene products from the D and the K end of the H-2 region are expressed on independent molecules or structures at the cell membrane.     

CELLS MEDIATING SPECIFIC IN VITRO CYTOTOXICITY : I. DETECTION OF RECEPTOR-BEARING LYMPHOCYTES

Spleen cells from mice immunized with allogeneic tumor cells are incubated on different fibroblast monolayers. The nonadsorbed cells are tested for cytotoxicity against ⁵¹Cr-labeled target cells. The cytotoxicity of nonadsorbed cells is much lower after incubation on fibroblasts syngeneic to the immunizing tumor cells than after incubation on fibroblasts syngeneic to the immune cells. This specific decrease of cytotoxic activity depends on the duration and temperature of incubation on monolayers. After incubation the monolayers are trypsinized and pure populations of adsorbed lymphocytes isolated by density gradient fractionation. The cytotoxicity of such trypsin-eluted, gradient-purified lymphocytes is much higher when these lymphocytes are isolated from fibroblasts syngeneic to the immunizing tumor cells than when they are isolated from fibroblasts syngeneic to the immune cells. These experiments demonstrate specific adsorption of immune cells onto fibroblasts carrying the immunizing antigens, and thus prove the existence of specific receptors at the surface of these immune cells. Spleen cells from mice immunized with two types of allogeneic tumor cells bearing different H-2 antigen alleles are incubated on different fibroblast monolayers. The results of such experiments show a differential specific adsorption pattern, suggesting independent adsorption of two populations of immune cells bearing receptors directed against either one or the other immunizing H-2 antigen. The existence of at least a majority of cells, each of which is homogeneous as to the specificity of its receptors, makes it likely that specific receptors are synthetized by the cells that bear them. The role of specific receptor-bearing cells in the killing process is discussed.     

Expression of alien H-2 and non-H-2 antigens on a murine mammary tumor cell line, MM46

Tumor-associated surface antigens on MM46, a transplantable ascites tumor from a spontaneous mammary carcinoma in a C3H/He mouse, were investigated. A complement-dependent microcytotoxicity test showed the expression of MM antigen, whose specificity appeared to be identical or cross-reactive with that of Ly-6.2 antigen, as reported previously. Moreover, MM46 was also found to express alien H-2 antigenic specificities, H-2.31 and H-2.28. H-2.31 represents the private specificity of the H-2Kd molecule, while H-2.28 represents a public specificity among molecules encoded by H-2K, H-2D, and H-2L loci in the majority of original H-2 haplotypes, including H-2d, but not H-2k, of inbred laboratory mice. The expression of MM antigen was found to be closely associated with the appearance of hypotetraploid tumor cells. These results suggest that alien H-2 and non-H-2 antigens appeared during ascites tumor cell conversion.     

SYNERGY DURING IN VITRO CYTOTOXIC ALLOGRAFT RESPONSES : I. EVIDENCE FOR CELL INTERACTION BETWEEN THYMOCYTES AND PERIPHERAL T CELLS

A mouse in vitro allograft system was used to evaluate the concept of T-T interaction in T cell-mediated cellular immunity. In analyzing the responsiveness of thymus-processed lymphocytes as obtained from different tissues, a heretogeneity within T cells was found in regard to their capacity to be immunized in vitro against transplantation antigens. Recirculating T cells were 10–20-fold superior to thymocytes, splenic T cells being intermediate. When few (1.5 x 10⁶) peripheral T cells, in numbers too small to yield good cytotoxic responses, were mixed with 14 x 10⁶ thymocytes and the cell mixture immunized in vitro against cell-bound alloantigens, cytotoxic activity was generated exceeding about 10–20-fold the values that could be explained by a pure additive effect. Synergy occurred also in a mixture of responder T cells derived from CBA (H-2^k) and AKR (H-2^k) mice. Thus AKR anti- C3H serum could be used for discriminating between thymus-derived and peripheral T cell-derived cytotoxic lymphocytes (CL). Cytotoxic activity produced during the synergistic interaction between thymocytes and peripheral T cells was about 70% T cell derived, the remainder being thymus derived. The synoptic interpretation of this finding and "limiting dilution" experiments of the responder cells suggested strongly that peripheral T cells provide the major source for precursor cells of CL, thymocytes acting mainly as helper (amplifier) cells.     

Kirsten murine sarcoma virus abolishes interferon gamma-induced class II but not class I major histocompatibility antigen expression in a murine fibroblast line

The effect of infecting fibroblasts with Kirsten murine sarcoma virus/murine leukemia virus (Ki-MSV/MLV) on constitutive and IFN-gamma-induced H-2 antigen expression was investigated. The fibroblasts used were two established cell lines (C3H10T1/2 and BALB/c3T3) and fresh embryo fibroblasts from C3H mice. Class I antigens were expressed constitutively by BALB/c3T3; infection with MLV, MSV or the two together had little effect on this constitutive expression. Class I antigens (H-2K, H-2D) were strongly induced on all three types of fibroblast by rIFN-gamma, and infection had little effect on this. None of the fibroblasts expressed constitutively detectable levels of class II antigen; however, C3H10T1/2 fibroblasts could be induced for both H-2A and H-2E by IFN-gamma. Infection of C3H10T1/2 with helper-free Ki-MSV, or MSV together with MLV, completely abolished this induction of class II antigens, while infection with MLV alone had little effect, implying that the abolition of class II induction was due to genomic regions of Ki-MSV not shared with Ki-MLV, probably the v-Ki-ras gene.     

Genetic and cellular aspects of xenogeneic mixed leukocyte culture reaction

The nature of the antigens stimulating xenogeneic lymphocytes was studied using "primed LD typing". Human lymphocytes were sensitized in vitro against mouse spleen cells and restimulated with spleen cells of mouse strains sharing non-H-2 antigens or various regions of H-2 with the initial stimulating strain. The largest thymidine uptake was caused by restimulation with cells from the specific primary stimulator or an H-2-identical strain. Species-specific antigens or strain-specific antigens carried in the C57BL/10 background account for less than 15% of the total stimulation; a non-H-2 antigen associated with the Mlsalpha genotype caused moderate restimulation, amounting to 25% of the average H-2 response. Within H-2, the strongest restimulation was caused by antigens controlled by the I-A subregion; the K and D regions caused moderate, the I-C and S regions very weak, and the I-B subregion no restimulation. Thus, the genetic control of antigens stimulating xenogeneic and allogeneic MLC responses requires T cells and adherent cells, but in the human-mouse MLC, both cell types must come from the human responder; the majority of the proliferating cells are T cells. It is suggested that allograft and xenograft reactions are fundamentally identical processes, and that the relative vigor of alloaggression may be explained by secondary potentiating mechanisms depending on species-specific interactions between aggressor and target cells.     

THE EXPRESSION OF HISTOCOMPATIBILITY ANTIGENS ON CELLULAR AND SUBCELLULAR MEMBRANES

The mouse isoantigens determined at the major histocompatibility locus known as H-2 have been found to be closely associated with the cellular surface membranes, with the membranes of the endoplasmic reticulum, and probably with those of the lysosomes as well. Mitochondrial membranes, on the other hand, show little or no H-2 antigen activity. Membrane material prepared from certain tissues, including brain and muscle, have no detectable H-2 antigenic activity. Evidence is presented which indicates that all of the H-2 antigens of the genome are expressed as a unit, supporting the hypothesis that the complex H-2 genetic locus consists of a single cistron. It is postulated that these histocompatibility antigens form some structural or functional unit in the membranes of cells.     

SPECIFIC CARCINOEMBRYONIC ANTIGENS OF THE HUMAN DIGESTIVE SYSTEM

A wide variety of human adult and fetal tissues were studied by immune-diffusion techniques in agar gel to determine whether they contained the tumor-specific antigen(s) previously found in coionic cancers. In the adult tissues it was demonstrated that identical antigens were present in all tested specimens of malignant tumors of the entodermally derived epithelium of the gastrointestinal tract and pancreas, but were absent from all other tested adult tissues. The common antigenic constituents, therefore, represent system-specific cancer antigens of the human digestive system. System-specific cancer antigens have not previously been demonstrated in humans. Experiments with fetal tissues demonstrated that identical antigens were also present in fetal gut, liver, and pancreas between 2 and 6 months of gestation. These components were named "carcinoembryonic" antigens of the human digestive system. On the basis of the present findings and the recent work regarding control of the expression of genetic potentialities in various types of cells, it was concluded that the carcinoembryonic antigens represent cellular constituents which are repressed during the course of differentiation of the normal digestive system epithelium and reappear in the corresponding malignant cells by a process of derepressive-dedifferentiation.