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# An approach to the mapping of antigens on the cell surface

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... More than 20 specificities are determined by the H-2 locus (which appears to comprise a series of closely linked genes in the 9th linkage group), and mice can be classified into 16 or more phenotypes on the basis of their constellations of H-2 antigens. The H-2 locus comprises at least two genetic regions separable by crossing over (the "D" and the "K" end) and determining antigens that are on different molecules (12,13), and are situated on different sites on the surface membrane (14,15). 8 angg~,~s (16): All mice have one or the other allelic form of this alloantigen. ...
... Thus, TL antibody cannot be absorbed by normal cells other than thymocytes, nor can TL antigen be detected by immunoferritin on cells other than thymocytes. The "blocking" method of mapping antigens on cell surfaces (14) has indicated that on thymocytes 0 occupies extensive areas adjacent to smaller areas, like islands, occupied by several other aUoantigens; ...
... One could consider more complicated models, but they hardly seem called for on present evidence. We have recently proposed a rudimentary map for the placement of alloantigens, including TL, H-2, and 0, on the surface of mouse thymocytes (14). Visual confirmation of this by electron microscopy in conjunction with labeled antibody would be an important step in confirming that the cell surface has inherent supramolecular conformation. ...
Article
The representation of mouse alloantigens belonging to three systems, H-2, θ and TL, on the surface of cells from thymus, spleen, lymph nodes, and peritoneal cavity, was studied by electron microscopy with ferritin-labeled antibody. As expected from earlier serological data, TL was confined to thymocytes, θ was found on thymocytes and lymphocytes, and H-2 occurred to some extent on all cell types observed. On reticular cells, lymphocytes, plasma cells, and eosinophils, the majority of the cell surface was occupied by H-2; thymocytes had considerably less H-2, and erythrocytes and peritoneal macrophages least of all. In every instance the representation of antigen was discontinuous, the fraction of the cell surface covered being characteristic both of the antigen and of the type of cell. H-2 and θ provide a striking example of this; H-2 is present in far higher amounts on lymphocytes than on thymocytes, whereas the converse is true of θ. Within areas positive for H-2 or θ, protuberances of the surface membrane were often antigen-negative. A better definition of cell surface structure, gained from studies such as this, is necessary for further inquiry into how the cell surface is assembled, and into selective gene action in relation to cellular differentiation.
... More than 20 specificities are determined by the H-2 locus (which appears to comprise a series of closely linked genes in the 9th linkage group), and mice can be classified into 16 or more phenotypes on the basis of their constellations of H-2 antigens. The H-2 locus comprises at least two genetic regions separable by crossing over (the "D" and the "K" end) and determining antigens that are on different molecules (12,13), and are situated on different sites on the surface membrane (14,15). 8 angg~,~s (16): All mice have one or the other allelic form of this alloantigen. ...
... One could consider more complicated models, but they hardly seem called for on present evidence. We have recently proposed a rudimentary map for the placement of alloantigens, including TL, H-2, and 0, on the surface of mouse thymocytes (14). Visual confirmation of this by electron microscopy in conjunction with labeled antibody would be an important step in confirming that the cell surface has inherent supramolecular conformation. ...
... Thus, TL antibody cannot be absorbed by normal cells other than thymocytes, nor can TL antigen be detected by immunoferritin on cells other than thymocytes. The "blocking" method of mapping antigens on cell surfaces(14) has indicated that on thymocytes 0 occupies extensive areas adjacent to smaller areas, like islands, occupied by several other aUoantigens;FIa. 3. Occurrence of H-2 alloantigens on nonlymphoid cells. ...
Article
The representation of mouse alloantigens belonging to three systems, H-2, θ and TL, on the surface of cells from thymus, spleen, lymph nodes, and peritoneal cavity, was studied by electron microscopy with ferritin-labeled antibody. As expected from earlier serological data, TL was confined to thymocytes, θ was found on thymocytes and lymphocytes, and H-2 occurred to some extent on all cell types observed. On reticular cells, lymphocytes, plasma cells, and eosinophils, the majority of the cell surface was occupied by H-2; thymocytes had considerably less H-2, and erythrocytes and peritoneal macrophages least of all. In every instance the representation of antigen was discontinuous, the fraction of the cell surface covered being characteristic both of the antigen and of the type of cell. H-2 and θ provide a striking example of this; H-2 is present in far higher amounts on lymphocytes than on thymocytes, whereas the converse is true of θ. Within areas positive for H-2 or θ, protuberances of the surface membrane were often antigen-negative. A better definition of cell surface structure, gained from studies such as this, is necessary for further inquiry into how the cell surface is assembled, and into selective gene action in relation to cellular differentiation.
... At least two possibilities may account for suppression of the response to noncoated determinants. First, there may be steric hindrance brought about by antibody coating determinants when the noncoated determinants are on the same molecule or on separate but structurally closely related molecules (23,24). Second, since RBC from B2/B 2 homozygotes presumably have twice as many determinants available for coating by anti-B2 as do RBC from B2/B 3 heterozygotes, the additional quantity of absorbed antibody may be sufficient to result in net suppression. ...
... Perhaps the A, B, and Rh antigenic sites have a spatial relationship such that maternal isoantibody combining with A and/or B suppresses, by means of steric hindrance, the immune response to Rh. That significant steric effects because of antibody may exist between antigens determined by different genetic systems of mice (products of the TL system and the "D" end of the H-2 system) has been shown in in vitro studies (24). Other studies, in addition to the present findings, suggest that in certain situations antibody to one or more determinants may suppress the immune respense to other determinants which are physically closely associated (30,31). ...
Article
The isoimmune response of fowl inoculated with RBC coated with antibody was investigated. Anti-B antiserum from a single animal was used to coat different donor type RBC. With each donor type RBC the immune response to the coated determinants is suppressed. Enhancement of the immune response to noncoated determinants occurs when they are products of an allelic gene or belong to a different blood group system. Coating some B antigen determinants suppresses the response to noncoated determinants of the same antigen, i.e., determinants which are products of the same B gene. Varying the quantity of passive antibody revealed that the degree of suppression and the degree of enhancement are negatively correlated. These findings support the concept that antibody-coated determinants function as carrier for noncoated determinants, provided a certain physical association exists between them. A further interpretation of these studies is that in certain situations an antibody to one antigen may interfere with events which lead to an immune response to a different antigen. The possibility, that the protection afforded by ABO incompatibility against Rh isoimmunization is because of a similar phenomenon, is discussed. A hypothesis is presented which states that where the immune response to certain antigens behaves as a dominantly inherited trait, and is associated with histocompatibility type, the nonresponder animals possess an antibody (perhaps cell bound) which interferes with the response to determinants for which it does not have specificity. Responders are assumed to lack this antibody because it has specificity for their major histocompatibility antigens.
... However, CD5 was discovered much earlier (late 1960s) with the description of the so-called mouse cell membrane allogenic determinants (CMADs) (Thy-1, Lyt-1, Lyt-2, Lyt-3, Lyt-4, Lyt-5, Lyt-6, etc.), which were used to distinguish different functional subpopulations of T and B lymphocytes. 4 In that context, the mouse Lyt-1 alloantigen (also known as Ly-A or Ly-1 and later known as CD5) emerged as one of the first clearly defined lymphocyte-specific antigenic systems. 5 This was achieved because of the generation of mouse alloantisera against irradiated murine leukemic T cells. 6 The characterization of these alloantisera allowed the initial identification of CD5 expression in T cells from thymus, spleen, and lymph nodes. ...
Article
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CD5 was one of the first surface receptors described for mouse and human T lymphocytes. Since then, it has been found to be highly expressed by regulatory T cells and a subpopulation of regulatory B cells, to be physically associated to the T and B cell antigen receptors, to negatively modulate TCR and BCR-mediated signals and to bind certain pathogen associated molecular patterns. These findings position CD5 as an attractive target for developing immunotherapies aimed at either boosting or dampening ongoing immune responses. Here, we will review available data on the function of CD5 and its involvement in regulation of immune responses in health and disease, as well as the evidence and future challenges for developing therapeutic strategies aimed at targeting CD5 for autoimmune diseases, cancer and infections.
... The analysis and mapping of the requisite plasma membrane constituents, "cell surface topochemistry," can be carried out with antigen or with a variety of lectins, monospecific reagents obtained from invertebrates, bacterial products, and viruses (38). The most widely applicable approach involves immunologic mapping of cell surface antigens (39,40). Old, Boyse, and their colleagues have identified six distinctive systems of antigens in the mouse thymus (41). ...
Article
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Young adult rat thymus and lymph node cell subpopulations were obtained by differential flotation on discontinuous BSA density gradients and assayed for properties characteristic of mature thymus-derived lymphocytes. One such subpopulation (C) of thymocytes was enriched in its ability to respond mitotically to a hemiallogeneic MLR stimulus, to localize in the parenchyma of lymph nodes and spleen, and to initiate a GVH reaction in a suitable host. These cells did not respond well to mitotic stimulation by PHA, they were lighter in density than the majority of mature lymph node thymus-derived lymphocytes, and they possessed a thymus-specific antigen (RTA) not present on peripheral lymphoid cells. We conclude that the acquisition of peripheral properties occurs sequentially, during an intrathymic differentiation cycle or shortly after the cells leave the thymus.
... Alloantisera could block the function of these IR gene-controlled substances by reacting with histocompatibility antigens which are physically close to the IR gene product on the cell surface. A precedent for the linkage of determinants both on the chromosome and on the cell surface is available in the mouse in the case of the H-2 and TL determinants (33). It is also possible that the alloantisera may contain antibodies directly reactive with and capable of directly blocking the specific IR gene products which are, in this instance, different from specific histocompatibility antigens. ...
Article
A number of autosomal dominant immune response (IR) genes have been identified in both mice and guinea pigs. These IR genes have been shown to be linked to the major histocompatibility antigens of the species and to be functionally expressed primarily in T lymphocytes. In order to more fully understand the relationship between IR genes, histocompatibility antigens, and immune recognition, the effect of specific alloantisera on lymphocyte stimulation induced by antigens under control of IR genes was examined. Using lymphocytes from strain 2 or strain 13 animals, the in vitro proliferative responses both to antigens which are known to be under genetic control (DNP-GL in strain 2 guinea pigs and GT in strain 13 guinea pigs) and to an antigen which is not known to be under genetic control (PPD) were inhibited to a similar degree and to a much greater extent than the response to phytohemagglutinin. However, when cells from F1 (2 x 13) animals are used, the alloantisera markedly inhibit only the response which is linked to the histocompatibility antigens against which the serum is directed. Thus, the anti-2 serum inhibited the response to DNP-GL but not to GT; the anti-13 serum inhibited the response to GT but did not affect DNP-GL response. The inhibitory activity of the alloantisera could not be removed by absorption with gamma globulin of the opposite strain. It can be concluded from these observations that immune response genes produce a cell surface-associated product and that this product plays a role in the mechanism of antigen recognition by the T lymphocyte. The mechanisms by which alloantisera block this process of antigenic recognition is not resolved nor is the relationship between the IR gene product and the antigen-binding receptor of the T lymphocyte. The approach described here offers a powerful tool for the resolution of these problems.
... The immunoglobulin molecules appear to be distributed on the surface of lymphocytes in discrete spots or patches in a way similar to that of various membrane components, like the histocompatibility antigens, the TL antigen, and others (15,21,22). In this respect our findings are different from those reported by Raft et al. (4) on normal mouse lymphocytes, and by Johansson and Klein (23) on human lymphocytes in cases of lymphoid leukemia; these authors have seen, by immunofluorescence, the immunoglobulins distributed as polar caps or half-moons. ...
Article
Small and medium lymphocytes from the peripheral blood and lymphoid tissues of the rabbit react in suspension with antibodies directed against different immunoglobulin determinants. Through immunofluorescence, it was possible to show that numerous discrete spots on the surface of the positive lymphocytes carry immunoglobulin molecules. The positive lymphocytes are about one-half of all lymphocytes in the different preparations; thymus lymphocytes are all negative. With antisera specific for rabbit IgM as well as with antisera directed against allotypic determinants specific for IgM or IgG, it was possible to show that about nine-tenths of the immunoglobulin-positive lymphocytes carry IgM molecules on their surface. With antisera directed against a- and b-locus determinants, it was also possible to demonstrate that both heavy and light chains were present in the surface immunoglobulins. Furthermore, in animals which were heterozygous at the a or the b locus, it was found that each lymphocyte had immunoglobulins synthesized under the influence of only one of two alleles. A very small proportion of lymphocytes could be shown to have a specific surface reaction with one antigen (horse ferritin); the proportion of these cells increased very much after immunization.
... CD5 was first discovered in the late 1960s and was known as Lyt-1, Ly-A, or Ly-1 (mouse) and T1 or Leu-1 (human) (1). It was initially used to distinguish subpopulations of T and B cells from other immune cells (2,3). ...
Article
Full-text available
CD5, a member of the scavenger receptor cysteine-rich superfamily, is a marker for T cells and a subset of B cells (B1a). CD5 associates with T-cell and B-cell receptors and increased CD5 is an indication of B cell activation. In tumor-infiltrating lymphocytes (TILs) isolated from lung cancer patients, CD5 levels were negatively correlated with anti-tumor activity and tumor‐mediated activation-induced T cell death, suggesting that CD5 could impair activation of anti-tumor T cells. We determined CD5 levels in T cell subsets in different organs in mice bearing syngeneic 4T1 breast tumor homografts and assessed the relationship between CD5 and increased T cell activation and effector function by flow cytometry. We report that T cell CD5 levels were higher in CD4 ⁺ T cells than in CD8 ⁺ T cells in 4T1 tumor-bearing mice, and that high CD5 levels on CD4 ⁺ T cells were maintained in peripheral organs (spleen and lymph nodes). However, both CD4 ⁺ and CD8 ⁺ T cells recruited to tumors had reduced CD5 compared to CD4 ⁺ and CD8 ⁺ T cells in peripheral organs. In addition, CD5 high /CD4 ⁺ T cells and CD5 high /CD8 ⁺ T cells from peripheral organs exhibited higher levels of activation and associated effector function compared to CD5 low /CD4 ⁺ T cell and CD5 low /CD8 ⁺ T cell from the same organs. Interestingly, CD8 ⁺ T cells among TILs and downregulated CD5 were activated to a higher level, with concomitantly increased effector function markers, than CD8 ⁺ /CD5 high TILs. Thus, differential CD5 levels among T cells in tumors and lymphoid organs can be associated with different levels of T cell activation and effector function, suggesting that CD5 may be a therapeutic target for immunotherapeutic activation in cancer therapy.
... El primer paso para la identificación y separación de las subpoblaciones de linfocitos T provino de la observación de que estas células expresaban glicoproteínas en su superficie ( 6 ) . Moretta en 1976, informó la presencia de receptores de superficie en los linfocitos exclusivos para la porción Fc de la inmunoglobulina G y de la inmunoglobulina M. Estas dos subpoblaciones fueron denominadas TM (o T p ) y T G (o T Y ) respectivamente, y se demostró que desempañaban diferentes actividades funcionales in vitro (7.8). ...
Article
La función de los linfocitos era desconocida hasta que Gowans en 1965 los identificó como células importantes del sistema inmune (1). En una revisión hecha por Trowell en 1958 (2), hace sólo 25 años, los linfocitos eran considerados como células que poseían únicamente atributos negativos y que a pesar de haber sido estudiados durante aproximadamente cien años, no había podido asignárseles función alguna.
Chapter
Many approaches to the study of the composition and molecular architecture of membranes require their isolation free of other cellular components. Several uncertainties inherent in such isolations must be considered in the interpretation of any experimental data. Some sources of ambiguity are as follows.
Article
Five methylcholanthrene-induced sarcomas were compared for their capacity to (a) absorb monospecific H-2 antisera, (b) induce tumor-specific immunity in syngeneic mice, and (c) metastasize early to the lungs. Comparison of the uptakes of monospecific H-2 antisera by the five different tumors showed that each of the tumors had a high, intermediate, or low surface representation of all of the seven specificities tested. No antigenic specificity was completely absent from any tumor, and no tumor had an unusually large or small amount of any individual specificity. The tumors could be placed in the sequence from one to five with respect to their H-2 antigenicity. The same five tumors were also ranked with respect to their capacity to induce a tumor-specific immune response in syngeneic mice. The tumor-specific immunogenicity had an inverse relationship to the H-2 antigenicity in that highly immunogenic tumors were those that had quantitatively less H-2 antigen on their surface and vice versa. Early metastases to the lung was associated with low levels of tumor-specific immunogenicity and high levels of H-2 antigenicity.
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antigen (H-2), isno longer detectable bythemethod ofinhibition ofimmunecytolysis. Infected cellsprelabeled with"4C-glucosamine alsoshowa correspondingly greater lossoftrichloroacetic acid-insoluble radioactivity than uninfected cells. Theloss ofH-2antigenic activity isnotduetotheviral inhibition of hostcell protein synthesis since cells cultured for18hrinthepresenceofcyclo- heximide havethesame amountofH-2activity asuntreated controls. Also, cells infected withencephalomyocarditis virus, a picornavirus, showno lossofH-2 activity ata timewhenhostcell protein synthesis iscompletely inhibited. VSV structural proteins associated invitro withuninfected L-cell plasma membranes do notrender H-2sites inaccessible totheassay.Although antibodies may notcombine withalltheH-2antigenic sites on theplasma membrane, anti-H-2 serumreacted withL cells before infection doesnotprevent a normal infection withVSV.H-2 activity canbedetected invirus samples purified fromthemediumofinfected L cells; this virus purified after being mixedwithL-cell homogenates showsgreater H-2activity thanvirus purified after being mixedwithHeLacell homogenates. However, VSVmadeinHeLacells shows no H-2activity whenmixedwithL-cell homogenates.
Article
Boyse and co-workers have identified several systems of murine isoantigens called Ly antigens (for lymphocyte antigens) (e.g. Ly-1, Ly-2, Ly-3, and Ly-5) which appear to be restricted to thymic lymphocytes and peripheral T lympho- cytes (1-3). Each system, defined by cytotoxic alloantisera, appears to be deter- mined by a genetic locus which contains two alternative alleles (e.g., Ly-2 a and Ly-2 b) governing expression of two alternative cell surface antigenic specificities (e.g. Ly-2.1 and Ly-2.2, respectively). TheLy-1 locus has been shown to reside in linkage group XII (4), and the Ly-2 and Ly-3 loci are extremely closely linked to each other in linkage group XI of the mouse (5). Using the antibody-blocking test (6), Boyse and co-workers have obtained evidence that the Ly-2 and Ly-3 antigenic determinants are topologically in close proximity on the cell surface. The close genetic and topological linkage of Ly-2 and Ly-3 have prompted Itakura et al. (5) to suggest that Ly-2 and Ly-3 may actually comprise a single complex locus, Ly-2,3, and that their respective antigenic determinants may reside upon a single molecule. Although the functions of the molecules bearing the Ly antigenic specificities (hereafter referred to as the Ly antigens) are unknown, recent evidence using the C57BL/6 inbred strain (phenotype Ly-1.2, Ly-2.2, and Ly-3.2) suggests that different functional subclasses of peripheral T lymphocytes may bear different sets of Ly antigens (7, 8). Peripheral T cells bearing only the Ly-l.2 specificity appear to perform a helper function in the humoral and perhaps the cellular immune response. Killer cells or their precursors and suppressor T cells appear to bear both the Ly-2.2 and Ly-3.2 specificities but not Ly-l.2. The majority of peripheral T-cells appear to bear all three specificities (Ly-l.2, Ly-2.2, and Ly- 3.2) and may be precursors of the functional subclasses bearing a restricted selection of Ly antigens. A genetic marker in the V region of mouse immunoglobulin L chains (called the I~-peptide marker) (9) has been demonstrated to be very closely linked to the * Support for this research was provided by a U. S. Public Health Service Research Grant (CA 15808) from the National Cancer Institute to P. D. G. and by a U. S. Public Health Service Research Grant (CA 14051) from the National Cancer Institute to the Massachusetts Institute of Technology Center for Cancer Research. $Fellow of the Leukemia Society of America. 476 Article Full-text available A new method for the detection of cell surface immunoglobulin labeled with isotopic precursors is described. The method consists of the aggregation of surface Ig on cells with specific antibody (heterologous) and the subsequent removal of antigen-antibody complexes by the combination of high speed centrifugation and immunoprecipitation of remaining soluble complexes using antibody to the heterologous Ig. Using this method, the kinetics of appearance of cell surface Ig and its turnover were studied in murine splenocytes. The results suggest that cell surface Ig is synthesized and transported in the same manner as secretory Ig rather than being synthesized on the plasma membrane. The turnover of intracellular and cell surface Ig in lymphocytes is slow. In contrast, intracellular Ig in plasma cells is rapidly secreted and usually without a cell surface phase. Cell surface Ig was shown to be radiolabeled with [3H]glucosamine, -galactose, and -fucose. The proportion of cell surface to intracellular (nonsurface) Ig labeled with these precursors suggests the same sequence of addition of sugars to Ig destined to be on the surface of lymphocytes as with Ig which will be secreted by plasma cells. Results with this new method also confirm earlier conclusions based on experiments using cell surface iodination: 8S IgM is the predominant Ig on the surface of murine splenocytes and the molecule appears to be attached by its µ-chains. Article 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-2b and H-2k 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. Article A brief overview is presented where the principal question is the possible contribution of protein dynamics to biological function. First the molecular enzyme kinetic model of the authors is presented, where the involvement of the mass-distribution of solvent-solute particles and of the environmental viscosity in enzyme action and regulation is outlined. Further subtitles deal with the functional role of dynamic displacement of cell membrane proteins, their relative proximity and the fluorescence methods for monitoring them, with special attention to that which was developed by the authors (flow cytometric energy transfer). Article Previous studies have shown that when a subline of plasmacytoma LPC-1 is grown in ascites form, the tumor cells harvested after 10–12 days (“late” cells) are resistant to lysis by cytotoxic T lymphocytes (CTL), apparently because the cell surface H-2-encoded molecules are blocked by a trypsin-sensitive glycoprotein of approximately 160 kdaltons. The glycoprotein (gp 160) is produced, and accumulates on the cell surface, as the growing cells enter the stationary phase. In contrast, LPC-1 cells harvested from the peritoneal cavity early in the transplantation cycle (ca. 4 days), while in exponential growth (“early” cells), have far less gp 160 and are susceptible to lysis by CTL. It is shown that, compared with early cells, the late LPC-1 cells are far less effective in adsorbing antibodies to products of the H-2 Kd and H-2 Dd alleles, but only slightly less effective in adsorbing antibodies to la antigen, and fully effective in adsorbing antibodies to several antigens not related to H-2 (e.g. PC.1, gp70, gp100, B220). The differences suggest that gp160 might preferentially block H-2-encoded molecules (especially K and D) Gp160 was also detected on another myeloma, MOPC 315 OPEC, a subline of MOPC 315, where it also appeared to block H-2 molecules but not PC.1. Additional evidence for an interaction between gp160 and H-2 molecules was obtained when sonicated membrane fragments were used to elicit secondary CTL responses to H-2 in culture. The response could be elicited by fragments from early but not from late LPC-1 cells, unless the late cells were treated with subtilisin (to remove gp 160) before sonication. Article Stepwise incubation with specific H-2 antibody and rabbit-anti-mouse Ig was used to modulate the expression of selected H-2 specificities on the surface of lymph node cells or EL 4 leukaemia cells. Modulation was reflected in a loss of sensitivity to complement-dependent lysis and a polar pattern of immunofluorescence (‘capping’). Specificity H-2.5 on H-2a cells was co-modulated with H-2.11 and so was H-2.11 with H-2.5 on H-2k cells. However, when H-2.11 on H-2k cells was modulated, the cells did not loose cytotoxic sensitivity for H-2.5. This asymmetry in co-modulation of H-2.5 and H-2.11 may be accommodated by the duplication model of the H-2 complex which postulates a double determination of H-2.5 in the H-2k (unlike H-2a) chromosome, with one determinant being in the K region (where also the H-2.11 determinant is located) and the other in H-2D. The modulation results with H-2.5 and H-2.11 are compatible with the view that various H-2K specificities may represent distinct antigenic sites on the same glycoprotein molecule whereas H-2D specificities are on separate molecule(s). The independent modulation of H-2D.4 and H-2K.11 specificities further indicates that molecules of the two classes are also secondarily not linked in the membrane structure. Modulation-induced cytotoxicity resistance was further shown to persist even when the specific H-2 antibody is newly added; this suggests that it is a change in the distribution of the H-2 antigen to be held responsible (rather than the loss of the sensitizing antibody). Article Many developmentally regulated membrane proteins of lymphocytes are ecto-enzymes, with their active sites on the external surface of the cell. These enzymes commonly have peptidase, phosphodiesterase or nucleotidase activity. Their biological roles are just beginning to be discovered. Although their expression is usually associated with particular stages of lymphoid differentiation, the same gene products are often expressed on the surface of certain non-lymphoid cell types outside the immune system, indicating that their functions cannot be unique to lymphocytes, nor can they be ubiquitous. The plasma cell membrane protein PC-1 (phosphodiesterase I; EC 3.1.4.1/nucleotide pyrophosphatase; EC 3.6.1.9), which was one of the first serological markers for lymphocyte subsets to be discovered, is a typical example. Within the immune system, PC-1 is confined to plasma cells, which represent about 0.1% of lymphocytes. However, PC-1 is also expressed on cells of the distal convoluted tubule of the kidney, chondrocytes, osteoblasts, epididymis and hepatocytes. Recent work has shown that PC-1 is a member of a multigene family of ecto-phosphodiesterases that currently has two other members, PD-1α (autotaxin) and PD-1β (B10). Within this family, the extracellular domains are highly conserved, especially around the active site. In contrast, the transmembrane and cytopiasmic domains are highly divergent. Individual members of the ecto-phosphodiesterase family have distinct patterns of distribution in different cell types, and even within the same cell. For example, PC-1 is present only on the basolateral surface of hepatocytes, while B10 (PD-1β) is confined to the apical surface. Analysis of conservation and differences in the sequence of their cytoplasmic tails may illuminate intracellular tar-getting signals. Ecto-phosphodiesterases may play a part in diverse activities in different tissues, including recycling of nucleotides. They may also regulate the concentration of pharmacologically active extracellular compounds such as adenosine or its derivatives and ceil motility. Some members may modulate local concentrations of pyrophosphate, and hence influence calcification in bone and cartilage. Article Staining en bloc in aqueous uranyl acetate following double fixation in glutaraldehyde and osmium tetroxide reveals two types of lymphocytes (dark and light) in most rat and mouse lymphatic tissues studied by transmission electron microscopy. While only dark lymphocytes populate the thymus, both dark and light lymphocytes congregate within the bone marrow and the thymus-independent areas or the spleen and lymph nodes. Two similar types of plasma cells coexist in the spleen and lymph nodes. Article Molecular recognition processes between cell surface elements are discussed with special reference to cell surface pattern formation of membrane-bound integral proteins. The existence, as detected by flow cytometric resonance energy transfer (Appendix), and significance of cell surface patterns involving the interleukin-2 receptor, the T-cell receptor–CD3 system, the intercellular adhesion molecule ICAM-1, and the major histocompatilibilty complex class I and II molecules in the plasma membrane of lymphocytes are described. The modulation of antigen presentation by transmembrane potential changes is discussed, and a general role of transmembrane potential changes, and therefore of icon channel activities, adduced as one of the major regulatory mechanisms of cell–cell communications. A general role in the mediation and regulation of intercellular interactions is suggested for cell-surface macromolecular patterns. The dynamic pattern of protein and lipid molecules in the plasma membrane is generated by the genetic code, but has a remarkable flexibility and may be one of the major instruments of accomodation and recognition processes at the cellular level. Article One question which is unresolved in developmental immunology is whether cortical thymocytes are the precursor cells which give rise to medullary thymocytes and peripheral T cells. Cortical thymocytes display a characteristic surface antigen phenotype (high TL and Thy-1, low H-2, no Qa-2, no Qa-3), are agglutinated by peanut agglutinin (PNA), and are unresponsive to concanavalin A (Con-A). The functionally more mature medullary thymocytes express a surface phenotype more closely resembling peripheral T cells (no TL, low Thy-1, high H-2, and some Qa-2), are not agglutinated by PNA, and are responsive to Con-A. An in vitro induction system has been devised in which mouse thymocytes undergo quantitative changes in surface antigens in less than 24 hr and increase their mitogen response to Con-A. The phenotypic changes are characterized by a decrease of TL and Thy-1 and an increase in H-2, Qa-2, and Qa-3. Studies in which thymocytes were fractionated on BSA gradients and by PNA agglutination demonstrate that the inducible cells have the properties of cortical thymocytes. Our data show that a subpopulation of cortical thymocytes can acquire phenotypic characteristics similar to medullary thymocytes and peripheral T cells. Article Hybrid antibody [F(ab')2] with dual specificity for mouse γG and ferritin was prepared from the corresponding rabbit antisera, providing a precise reagent for locating mouse γG on cell surfaces. Viable cells were exposed successively to (a) mouse antibody to a cell surface antigen, (b) the rabbit hybrid antibody, and (c) ferritin, before preparation for electron microscopy. This method of See PDF for Structure. labeling is sensitive and specific and clearly lends itself to the introduction of visual markers other than ferritin. Other advantages are uniformity of labeling, ease of purification of the reagent, and circumvention of the many drawbacks arising from coupling ferritin to antibody chemically. Chapter Immune responses begin with the binding of antigen to surface receptors on lymphocytes. In this reaction, called ‘antigen recognition’, lymphocyte receptors discriminate by variations in the strength of the binding reaction among vast numbers of antigens, including many that differ from each other only very slightly in structure. Antigen recognition by B lymphocytes is now well understood: their recognition molecules are cell surface antibodies (Warner, 1974) and antigen binding by antibodies has been characterized, for many antigens, at a highly sophisticated chemical level. However, antigen recognition by T cells (i.e. thymus-derived lymphocytes) is poorly understood and remains one of the most pressing challenges of modern immunology. Chapter The main topic of the 22nd Colloquium of the “Gesellschaft für Biologische Chemie” being the dynamics of cellular membranes, I should like to review some genetic aspects of surface antigens, with emphasis on those phenomena that may have some bearing on the dynamics of cell surfaces. When speaking of dynamics I am not referring to the biochemical turnover of surface components, but to the phenotypic changes in general that a somatic cell undergoes during its life span. It has been realized that surface antigens do not represent stable, unalterable phenotypic traits. In the following, I should like to discuss some factors responsible for such phenotypic changes. Chapter Wenn man vor die Aufgabe gestellt wird, den Stand unserer derzeitigen Kenntnisse über die Störungen der Immunantwort in einem Referat zusammenzufassen, so sieht man sich einer solchen Flut von kasuistischen Beobachtungen, experimentellen Resultaten und pathogenetischen Deutungsversuchen gegenüber, daß eine erschöpfende und allen Forschungsergebnissen gerecht werdende Darstellung der Sachverhalte unmöglich erscheint. Man ist somit gezwungen — unter Verzicht auf die Würdigung mancher grundlegenden Arbeiten und Prioritäten — einige wenige, für das Verständnis der Problematik essentielle Erkenntnisse herauszugreifen. Und was wäre vor diesem Auditorium naheliegender, als die wichtigsten klinischen Krankheitsbilder, die in den letzten 20 Jahren entdeckt wurden, Revue passieren zu lassen, jene seltenen, aber gut definierten primären Defektsyndrome vor allem, die als Extremfälle der zahlreichen Abstufungen und Spielarten menschlicher Immunopathien, die wesentlichen klinischen und immunbiologischen Gegebenheiten am deutlichsten veranschaulichen. Chapter Histocompatibility (H) antigens are genetically segregating cell surface markers which elicit an immune response by the host following the transplant of foreign tissues. Allografting thus furnishes an excellent tool for distinguishing these antigenic cell surface markers, as it causes the host to recognize a set of cellular antigens which distinguish him from the graft donor. Article It is almost normal practice to greet a newly described biological phenomenon by naming a substance by which one hopes it is mediated. Even if the phenomenon has been correctly described a simple mediator may not account for it; if incompletely or incorrectly described, search for a simple mediator is at best a hazard. Mediation may be by an array of components, e.g. like a chain of enzymes. Complexity mounts as we delve deeper into biological phenomena and their inter-relationships; there are the multiple components of complement, of coding, of capping, of clotting. Article The premise that complex carbohydrate structures have a functional role at the cellular level must be immediately followed by the admission that not a single mammalian cellular complex carbohydrate structure has yet been clearly associated with a physiological cellular function. On the other hand, there is now abundant reason to believe that such structure-function relationships will soon be demonstrated. It also seems clear that the rate of progress in this area is directly related to the rate of progress with the structural half of the relationship. At the moment, there are two general sources of structural information. First, detailed structural information on a fairly broad range of complex carbohydrates that are extracellular products of cells is now available. This information can be used to establish a conceptual framework of the kinds of structure to look for at the cellular or subcellular level. Second, there now exists detailed structural information on one or two cellular complex carbohydrates of unknown function (blood group substances). Article Interest in the Major Histocompatibility Complex (MHC) has grown such that it occupies a central position in immunobiology (Dorf, 1981). Many of the concepts developing which concern MHC biology extend in their relevance beyond the immune system. For example, an important aspect of MHC encoded proteins is their involvement in several cell-cell interactions in the immune response. The recognition of target cells by cytolytic T lymphocytes, collaboration between T and B lymphocytes in antibody production and antigen presentation by macrophages to T lymphocytes are regulated by MHC encoded cell-surface molecules. Thus, the identification of structural features of MHC encoded molecules important in such aspects of the immune response should have implications for cell-cell interactions in general. Article Long-term four month treatment with heterologous antilymphocyte serum (ALS) produced immunoincompetence of thymus, spleen and bone marrow lymphocytes as measured by their ability to produce a graft-versus-host reaction. Twenty-four hours following discontinuance of treatment this immunoincompetence is manifested by all three cell populations. Forty-eight hours following cessation of administration of ALS an immunocompetent population of lymphoid cells is demonstrable in the thymus gland. This activity is again confirmed at 168 hr, a time at which no immunocompetent cells are detectable in the spleen or bone marrow cell populations. Article In this study a variety of human lymphocytes of known B or T cell type, obtained from multiple sources, were prepared for scanning electron microscopy (SEM) by the critical point drying method. Distinction between normal B and T lymphocytes was relatively easy in most instances, on the basis of their surface architecture. Using immunological methods, between 20 and 30% of normal peripheral blood lymphocytes (PBL) were identified as B cells and from 69 to 82% as T cells. SEM results showed that 20% of the PBL had a complex villous surface and approximately 80% of cells were smaller and had a relatively smooth surface. Comparison of the above data and enrichment of B cells from PBL, by centrifugation after T cell rosettes had formed, indicated that the "villous" cells were B lymphocytes and the "relatively smooth" cells were T lymphocytes. T cells obtained from two human thymuses were also of the generally smooth cell type. Further evidence for the distinction of B and T lymphocytes, on the basis of surface morphology, was obtained from the examination of cultured lymphoid cell lines of known B or T cell derivation. Cells from cases of chronic lymphocytic leukemia also provided support for the above interpretations. Five of six untreated cases were clearly of B cell type by immunologic and SEM criteria. One unusual case showed the presence of T and B lymphocytes in almost equal numbers by SEM and a mixture of B and T cells by immunologic markers. An additional case that had received chemotherapy showed numerous atypical cells that were difficult to classify by SEM. Detailed examination of the smoother T cells showed that at least half of them had a moderate number of surface digitations and a small proportion had an intermediate surface morphology with a relatively large number of surface digitations. The latter presented difficulties in classification and may correspond to different stages of differentiation and represent subpopulations of lymphocytes. The distinction between human B and T lymphocytes on the basis of their surface architecture can be made by SEM of critical point dried samples, with relative ease in most but not all instances. The effects of stimulation, cell cycle, differentiation, intercellular contact, and density of cell population, on the surface architecture of lymphoid cells, remain to be determined. Chapter Faced with the familiar problems of morphogenesis in all its aspects, including its breakdown in cancer, we are more and more forced to conclude that the cell surface must embody elaborate codes of recognition which regulate the orderly deployment of cells within multicellular organisms. The task of elucidating the organization of cell surface elements in terms that might help us to comprehend this can profitably be tackled as an exercise in immunogenetics. This is because serology provides the only general method for recognizing components of the cell surface as individual, discrete elements. Conventional immunogenetic techniques for studying cell surface antigens are somewhat inadequate in this context, dominated as they have been by the practicalities of delineating intraspecies antigen variation (allo-antigens) based on genetic polymorphism (allelism), for purposes of blood transfusion and tissue transplantation. Some progress is being made with new applications directed to the topography or regional distribution of antigens on the cell surface. Article Human lymphocytes of known B or T derivation were examined by scanning electron microscopy (SEM) before and after rosetting with SRBC. After collection of the cells onto silver membranes the samples were prepared for SEM by the critical point drying method. Sheep RBC frequently underwent sphero-echinocyte transformation and multiple projections extended from their surfaces. This was readily noticeable after storage of SRBC in the cold and washing in Hanks, but more prominent after rosetting. These erythrocyte surface alterations were less apparent when freshly withdrawn cells were used. Spontaneous sheep erythrocyte rosettes (E-R), a marker for human T lymphocytes, were prepared with normal peripheral blood lymphocytes (PBL), thymic cells, and cultured T cells. EAC-rosettes (EAC-R), used to identify B lymphocytes with complement receptors, were prepared with normal PBL and cultured B cells. The majority of rosetting T lymphocytes had generally smooth surfaces while about 20% had an intermediate number of microvilli and 15% were more villous and indistinguishable from villous B cells. Studies of rosetting thymocytes and cultured T cells however indicated that the surface of some T cells alters on rosetting, becoming more villous and thus account for the higher numbers of villous T cells seen in E-rosettes. Point to point contact sites between SRBC and T lymphocytes were more frequent than broad zones of attachment. The majority of rosetting B lymphocytes had multiple microvilli, about 25% had a moderate number of microvilli and less than 10% had smooth surfaces similar to those of most T cells. Areas of contact between EAC and B lymphocytes were frequently broad zones of attachment. The study confirms that in many cases B and T lymphocytes can be distinguished by their surface architecture as seen under the SEM; however, about 20% of rosetting B and T cells have similar surfaces with intermediate numbers of surface microvilli and cannot be distinguished by SEM without parallel immunologic identification. Chapter The interaction of a cell with its extracellular environment occurs at the surface of its plasma membrane. The specialized functions of differentiated cells must, in some way, be correlated with the quality, quantity, and organization of the carbohydrate, lipid, and protein components of their membranes. The highly specialized immunological functions of mammalian cells are determined by specific chemical structures, cell surface antigens, and antigen receptor sites, which are responsible for the initiation of the immune response. The chemistry of several erythrocyte antigens has been elucidated in the last decade, but little or nothing is known about the chemistry of the majority of the antigenic sites on cell surfaces. Brief consideration is given here to the already classic studies on the dominant role of carbohydrates as the specific antigenic determinants of human blood groups and to the role of lipids as stabilizers for the antigenic determinants of membrane proteins. Prime consideration is given to studies on membrane proteins, which constitute an important class of antigenic determinants on cell surfaces. Chapter Human leukocyte antigens (HL-A) can be used to type an individual just as he can be typed by the ABO antigens on his erythrocytes. HL-A antigens are determined by the genes on a single pair of chromosomes (chromosome 6) that occupy a region defined as the HL-A complex. The HL-A gene complex can be divided into two regions, as is true of the genetic loci for the H-2 system in the mouse (Fig. 1). Each sublocus of human HL-A carries a series of alternative alleles that appear to be codominant. Within a family, a given sublocus 1 (LA) determinant will always travel with the same sublocus II (Four) determinant. An individual can have only two to four specificities, each of which is controlled by many alleles. To date, 14 LA and 26 Four antigens and their corresponding alleles have been identified. Table I lists some of these antigens and alleles; a more extensive listing is given by Kissmeyer-Nielsen et al (1972). Examples of the possible inheritance patterns of HL-A antigens are shown in Fig. 2. Chapter The concept of a plasma or cell membrane has evolved in little over a century from the simple nineteenth century idea of a passive bag containing the cell “sap” to the complex and dynamic subcellular fraction we think of today. The work of Moldenhauer in 1812 [cited in Bresnick and Schwartz (1969)] made possible the separation of tissues into component parts and the recognition of a limiting unit in the cell, i.e., a boundaryIt was not until the work of Nägeli in 1855 (Nageli and Cramer, 1855), however, that the presence of a separate structure formed from the proteinaceous material of the cell was conceptualizedHe first coined the term “Plasmamembran” and recognized that the membrane was responsible for conferring osmotic properties on the cell. Article Previous work with the antibody-blocking technique showed that the map of surface components for thymocytes prefixed with paraformaldehyde is the same as the map for unfixed thymocytes, with the following exception: after exposure to anti-TL or anti-Db, TL and H-2Db occupy adjacent positions on unfixed cells but not on fixed cells. This was interpreted as an indication that activation of particular components of the surface phenotype initiates ordered changes in the display of cell-surface molecules, approximation of TL and Db in this instance. These studies have now been extended to the H-Y component on the surface of male cells. On fixed male mouse thymocytes, H-Y lies adjacent to TL and relatively distant from H-2Db, H-2Kb, H-2Lb, Lyt-1.2, and Lyt-2.2. However, on unfixed male mouse thymocytes, similarly exposed to H-Y antibody, H-Y and H-2Db are adjacent. Presumably, this engagement of H-Y sites by H-Y antibody brings H-Y and H-2Db together. Evidence that this change in pattern may be physiologically relevant comes from the finding that testosterone, but not estradiol, caused the same selective approximation of H-Y and H-2Db. Article Rabbit antisera prepared against embryonic chick liver cells have been characterized using cell cytotoxicity tests, antibody absorption tests, inhibition of cell-cell reaggregation and cell-aggregate adhesion assays. The sera contain antibodies which specifically block embryonic chick liver cell-cell aggregation with no effect on other cell types or embryonic liver cells of other classes tested. Assays with sera absorbed with live cells, neural retina cells, or erythrocytes as well as with sera raised against these cells, indicate that this anti-aggregation effect is not merely the result of coating the cell surface with non-specific antibody. The anti-aggregation specificities of the sera have been checked by absorption experiments with a variety of membrane preparations. These results suggest significant cross-reactivity only between chick liver and kidney, perhaps reflecting the relatedness of the epithelial elements of these tissues. The immunoglobulin G fraction, F(ab')2 and monomeric fragments Fab' retain activity suggesting that the antisera inhibit cell-cell adhesion via blockage of cell surface ligands and not by cell surface modulation effects or cytotoxicity. The organ and class specificities of the antisera suggest that the inhibition of cell-cell aggregation is mediated by the direct binding of antibody to the embryonic chick liver cell adhesion ligand system. Article Mouse histocompatibility antigens were solubilized from lymphocyte membranes by limited papain degradation. Spleens from Balb/c mice (H-2d), enlarged by administration of lymphoma cells, were used for the membrane preparations. The solubilized protein components were purified by ion-exchange chromatography, gel filtration and discontinuous polyacrylamide-gel electrophoresis. A further characterization was achieved in a two-dimensional protein mapping system using disc-electrophoresis and isotachophoresis in the first and second dimension, respectively. The purification was monitored for alloantigens H-2.4(D) and H-3.21(K) (H-2D and H-2K represent two regions of the H-2 system). On Sephadex G-100 partially purified H-2-alloantigen chromatographed in a region of Mr= 35500. The pI of these substances showed a maximum at pH 5.5 and 5.3 for H-2.4 and at 5.2 for H-2.31. Free SH- and dithio-groups were determined with 5,5′-dithiobis(2-nitrobenzoic acid). 1.56 μmol SH/μmol alloantigenic material was found, but no SS-bond could be detected after alkaline cleavage. This suggests the absence of covalently linked protein subunits in papain-solubilized molecules bearing single private specificites. This was confirmed after reduction and alkylation in 4 M urea: 30–50% of the alloantigenic activity was retained and shown, in polyacrylamide-gel electrophoresis, to migrate in the original region of activity (RBPB 0.29 and 0.33 for H-2.4; about 0.33 for H-2.31, BPB = bromophenol blue). After reductive alkylation gel filtration resulted in a further purification of the alloantigenic material. Highly purified substances showed a diffuse staining pattern and a broad serological profile after electrophoresis. We assume this heterogenity to be a genuine property of H-2 antigens. Structural and genetic implications of these findings are discussed. Article 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-2b, H-2a, and H-2k). 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. Article Mouse H-2 histocompatibility antigen has been extracted, solubilized, and partly purified from the cells of an A strain spontaneous leukemia carrying TL (thymus-leukemia) antigens. H-2 and TL. 1, 2, 3 activities were measured by inhibition of the cytotoxic effect of the corresponding isoantibodies. TL activity was associated with the H-2 active fraction obtained by solubilization and fractionation by gel filtration. TL specificity was largely separated from H-2 antigen by subsequent chromatography on DEAE Sephadex as an adjacent component in a series of fractions. The soluble H-2 antigen prepared from the leukemia cells was tested for most of the specificities determined by H-2a with no exceptional results. TL. 1, 2, 3 activities, measured as each component separately, were located in approximately the same position; there is no clear indication yet whether the three TL specificities are separable from one another. It appears that in addition to the close genetic linkage between the H-2 and TL loci, and their reciprocal interaction in producing H-2 and TL antigens, these antigens exhibit some similarity at the chemical level. Article 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-2b, H-2a, and H-2k). 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. Article 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. Article An immunofluorescent technique is described which permits the locali-zation and the semi-quantitative evaluation of isoantigens. Studies of normal and tumour cells have demonstrated that H-2 isoantigens are located in discrete areas on the cell surface. The thymus of 20-25-day-old mice was found to contain approx-imately 85 per cent lymphoid cells with a very low isoantigen content. These cells are considered to represent thymus cortical lymphoid cells. Cortisol treatment of mice reduced the relative number ofcells with low isoantigen content to 10 per cent. Two systems of isoantigens confined to thymocytes and lymphocytes have been defined in the mouse by cytotoxic isoantisera. The two genetic loci have been designated Ly-A and Ly-B. Typing of 25 mouse strains and sublines indicates that each system comprises only 2 alleles, determining alternative isoantigens Ly-A. 1 or Ly-A. 2, and Ly-B. 1 or Ly-B. 2, respectively. There is no evidence of multiple alleles or that either locus is compound. Tests for genetic linkage were performed by serological typing of mice from segregating generations. None of the four loci H-2 (group IX),$\theta $, Ly-A and Ly-B is closely linked to any other of the four. Ly antigens are found in high concentration on thymocytes and in lesser amounts on lymphocytes. The small absorptive capacity of bone marrow cell suspensions is probably due to the presence of mature lymphocytes rather than thymocyte-lymphocyte precursors, which according to preliminary evidence lack Ly antigens. Ly-B. 1 is exceptional in that the disparity between thymocytes and lymphocytes in content of antigen is greater than that of the three other antigens. When Ly antiserum is injected into mice of the relevant Ly type, under conditions which give complete absorption of H-2 antibody in vivo, Ly antibody is not extensively absorbed; this is to be expected from the limited tissue distribution of Ly antigens. Absorption of Ly and$\theta $iosantibodies in vivo is reduced by treatment of the recipients with cortisone or lethal total-body irradiation. The reduction in titre of Ly and$\theta $antibodies in such mice may be no more than that resulting from dilution. Absorption of injected H-2 antibody also may be reduced by treatment of the recipients with cortisone but in this case the effect of cortisone does not approach the virtual abolition of absorption that is seen with Ly and$\theta $antibodies. The thymocytes and lymphocytes of chimeras formed by restoring lethally irradiated mice with allogeneic bone marrow have the Ly-A type, Ly-B type and$\theta $type of the donor. The content of Ly antigens on cells of different leukaemias varies widely and shows no correlation with presence or absence of TL (thymus-leukaemia) antigen; TL+ leukaemias may be Ly- or Ly+ and TL- leukaemias may be Ly- or Ly+. Five systems of isoantigens are now demonstrable on mouse thymocytes by means of cytotoxic isoantisera. Of these, TL is an exclusively thymic antigen. Ly-A and Ly-B antigens occur also on lymphocytes.$\theta \$ occurs on thymocytes, on lymphocytes, and in brain. The fifth antigen, H-2, is still more widely distributed and differs from the other four in being represented less strongly on thymocytes than on lymphocytes.
Article
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 degrees 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 degrees 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.
Article
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.
Article
MUCH work has been done on experimental auto-immunization with nervous tissues, and the relation of the resulting pathology to that found in demyelinative diseases of the human nervous system1. Furthermore, the existence of organ-specific lipid haptens has long been recognized; recently, Joffe, Rapport and Graf have identified one of these haptens as a galactocerebroside2. No strain-specific iso-antigens were described by these authors1,2.
Article
Old, Boyse and Stockert reported the presence of a common iso-antigen (LT) in certain leukaemias of two strains of mice, and in thymus of only one of these strains and one unrelated strain1. Later, the LT iso-antigen was found in normal thymus of eight out of twenty-four strains2. The LT iso-antigen was not detected in the normal tissues examined other than thymus1,2.
Article
Mouse H-2 histocompatibility antigen has been extracted, solubilized, and partly purified from the cells of an A strain spontaneous leukemia carrying TL (thymus-leukemia) antigens. H-2 and TL. 1, 2, 3 activities were measured by inhibition of the cytotoxic effect of the corresponding isoantibodies. TL activity was associated with the H-2 active fraction obtained by solubilization and fractionation by gel filtration. TL specificity was largely separated from H-2 antigen by subsequent chromatography on DEAE Sephadex as an adjacent component in a series of fractions. The soluble H-2 antigen prepared from the leukemia cells was tested for most of the specificities determined by H-2(a) with no exceptional results. TL. 1, 2, 3 activities, measured as each component separately, were located in approximately the same position; there is no clear indication yet whether the three TL specificities are separable from one another. It appears that in addition to the close genetic linkage between the H-2 and TL loci, and their reciprocal interaction in producing H-2 and TL antigens, these antigens exhibit some similarity at the chemical level.
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In the ABO system the genes control the arrangement of sugar residues that determines blood-group specificity.
Article
A clear-cut serological differentiation between AKR lymphocytes of thymic and non-thymic origin is reported: these two cell types are antigenically distinct. In newborn mice, the AKR thymic antigen was found at a high concentration only in thymus. In adult mice, the antigen was present at a high level in thymus, all nervous tissues tested, and some leukemias. It was present at much lower levels in lymph node lymphocytes, splenic lymphocytes, appendix, lung, and certain other leukemias, which appeared to be of non-thymic origin. The AKR thymic antigen was present at a high level in thymus and nervous tissues of RF mice, but was absent from thymocytes of sixteen other mouse strains. These sixteen strains possessed the C3HeB/Fe thymic antigen. The distribution of this antigen in neonatal and adult tissues of the strains tested was similar to that of the AKR thymic antigen in AKR mice. No exceptions were found. These results were obtained by use of immune cytolysis, and of a new method for the quantitative treatment of data from absorption experiments.
• A Shimada
• S G Nathenson Watkins
8 Shimada, A., and S. G. Nathenson, Biodhem. Biophys. Res. Commun., 29, 828 (1967). 9 Watkins, W. M.. Science, 152, 172 (1966).