Lymphoid cells differ in the constitution of their surface membranes as demonstrated by study of their surface antigens. Aside from the surface immunoglobulin (sIg), 1 which has been demonstrated on a proportion of lymphoid cells (1, 2), some antigenic determinants of the cell membranes appear to be specifically related to the pathway of differentiation followed by the cells which bear them. These "differentiation antigens" (3) can thus be used to recognize, among lymphocytes, those which are derived from the thymus, or T lymphocytes, and those which are thymus independent, or B lympbocytes. In the mouse, alloantisera raised against the 0 antigen have been widely used for the recognition of T lymphocytes (4). Heteroantisera can also be obtained by immunmation of other species with mouse thymocytes (5) or brain (6); after appropriate absorption on mouse tissues, in vivo (5) or in vitro (6), these antisera recognize antigenic determinants which appear to be present only on T lymphocytes and which have been called "mouse-specific lymphocyte antigens," MSLA (5), brain-associated 0 antigen, BAe (6). It is also possible to obtain, by immunization with mouse lymphoid cells depleted in T cells, heteroantisera which after absorption by mouse thymocytes appear to recognize antigenic determinants present on B but not on T lymphocytes, and called for this reason "mouse-specific bone marrow-derived lymphocyte antigen(s)," MBLA (7).

The gel filtration behavior, in the presence of detergents, of membrane-bound IgM from normal mouse spleen B lymphocytes was compared to that of secretory IgM from mouse plasma cells. The proteins were labeled either by surface radioiodination or biosynthetically with radioactive amino acids. Cell lysates were fractionated on calibrated Sepharose 6B columns in the presence of the detergents Nonidet P40 or deoxycholate. Eluted fractions were immunoprecipitated and the reduced or unreduced precipitates were analyzed by sodium dodecyl sulfate gel electrophoresis followed by radioautography. Surface 125I-labeled 8S IgM exhibited a gel filtration pattern in Nonidet P40 corresponding to much higher apparent molecular weight than that of secretory 8S IgM, a difference that almost disappeared when gel filtration was performed in the presence of deoxycholate, which forms much smaller micelles than does Nonidet P40. Biosynthetically labeled lymphocytes contain two types of IgM molecules differing in their gel filtration behavior and fate: one identical to secretory 8S IgM of plasma cells and secreted in the medium during chase periods, and the other identical to surface 25I-labeled IgM and remaining cell-associated. Because the surface-bound 8S IFM was not found to be associated with other labeled molecuJes, it is likely that the detergent-binding behavior of surface IgM is due to a hydrophobic segment carried by these Ig molecules. That lymphocytes synthesize two types of ju chains was also shown by the use of tunicamycin, an inhibitor of glycosylation. In its presence, two unglycosylated y chains were observed: one identical in size to that made by tunicamycin-treated plasma cells, and the second slightly larger. Gel filtration in Nonidet P40 of the cell lysates of tunicamycin-treated lymphocytes showed that the nonsecretory 8S IgM contains this second type of u chains, whereas the IgM mo les of the secretory type contain plasma cell-like ,g chains. It is suggested that membrane IgM us chains contain a hydrophobic segment which is responsible for its association to the membrane.

Abstract. Free and membrane-bound ribosomes have been isolated from a mouse plasma cell tumor secreting immunoglobulins, and allowed to synthesize proteins under cell-free conditions. Analysis of the products of synthesis by autoradiography of immunoelectrophoresis and peptide maps indicates that both types of ribosomes synthesize heavy and light immunoglobulin chains.Introduction. Cells that synthesize large amounts of proteins intended for secretion are characterized by a well-developed endoplasmic reticulum to which are attached numerous ribosomes, called membrane-bound (MB) ribosomes in opposition to ribosomes found free in the cytoplasm (free ribosomes).On the basis of biochemical evidence obtained with pancreatic tissue, Siekevitz and Palade have proposed that the MB ribosomes are the site of synthesis of secretory proteins, and that these proteins are subsequently transported into the endoplasmic reticulum cisternae.1 Observations made with several other tissues have confirmed these concepts, and the link between MB ribosomes, endoplasmic reticulum membranes, and the synthesis and transport of secretory

The ultrastructural features of B-, T-, and surface Ig(sIg)-bearing cells have been studied on cell suspensions from lymphoid organs of mice at different stages of immunization. The cells were identified by exposure to rabbit antibodies against mouse-specific lymphocyte antigens (MSLA) or brain-associated θ antigen (BAθ) for T cells, mouse-specific bone marrow-derived lymphocyte antigens (MBLA) for B cells, and mouse Ig for sIg-bearing cells. The rabbit antibodies fixed on the cell surfaces were detected by peroxidase-labeled sheep anti-rabbit Ig antibodies or by a "bridge" technique using southern bean mosaic virus or bacteriophage T4 as the final markers. In some experiments, short-lived lymphoid cells were labeled in vivo with repeated tritiated thymidine and the ultrastructural detection of their surface antigens was combined with radioautography. MBLA+ lymphoid cells showed a whole range of ultrastructural patterns. Most were small and medium-sized lymphocytes with a clear cytoplasm containing mono- and polyribosomes, but they comprised also blasts and large cells with various amounts of endoplasmic reticulum, as well as plasma cells at different stages of maturation. sIg-bearing cells appeared to be identical with MBLA+ cells, except that sIg was less easily detectable on large blasts, and only very rarely observed on plasma cells. MSLA+ and BAθ+ cells fell into three categories. One of them (T1 cells) consisted of small to medium-sized lymphocytes with a clear cytoplasm and few organelles, mostly monoribosomes. A second consisted of large cells (T2 cells) characterized by numerous polyribosomes often in a "rosette"-like pattern, occasional dark, membrane-bound granules, and a developing "filamentous network." The third, very characteristic type, (T3 cells) was represented by dark small to medium-sized lymphocytes, usually containing large amounts of closely packed ribosomes and showing a striking accumulation of filamentous network, often condensed in large areas devoid of cell organelles. This filamentous network appeared to correspond to the cytochalasin B-sensitive system of microfilaments found in other cells and considered to be one of the contractile elements of the cell. The T3 lymphocytes showed frequently vesicles suggestive of a strong pinocytic activity, and assumed a variety of shapes, including uropods. Evidence is presented that T1 lymphocytes represent "virgin" T cells, T2 "activated," and T3 "differentiated" lymphocytes.

In the present work, we attempted to detect immunoglobulin on or in thymocytes, various thymus-derived lymphocytes, spleen cells, and B lymphocytes, using cell-surface radioiodination as well as biosynthetic labeling procedures. The labeled proteins were precipitated by antibodies directed against mouse immunoglobulin chains and the precipitates were analyzed by radioautography after Na dodecyl sulfategel electrophoresis (9), a sensitive procedure with high resolving power. MATERIALS AND METHODSLactoperoxidase-Catalyzed Radioiodination of Cell-Surface Proteins. Thymocytes and spleen-cell suspensions were prepared as described (3), and used only when more than 95% of the cells were viable, as judged by trypan blue exclusion. Iodination was performed by the method of Marchalonis et al. (10) usually on 108 cells with 1 mCi of carrier-free Na-125I (80-140 mCi/ml, Amersham), 100 ,g of lactoperoxidase (Sigma, St. Louis, Mo.), and 30 ,l of 8.8 mM H202. After a 5-min incubation at 300, the cells were centrifuged at 4°. They were washed once with 5 mM L-cysteine -HCl in phosphatebuffered saline (pH 7.4), twice with 5 mM L-cysteine-HCl-10 mM KI in phosphate-buffered saline, and once with 10 mM KI in phosphate-buffered saline. Trypan blue exclusion showed no loss of viability. Three procedures were used to solubilize the labeled cell-surface proteins. Bethesda, containing 5% fetal-calf serum), followed by extensive dialysis against phosphate-buffered saline of the culture medium containing released labeled proteins. The extracts obtained after the various procedures were analyzed for total, as well as trichloroacetic acid-precipitable, radioactivity with a Packard Autogamma spectrometer. Usually 10-30% of the radioactivity was not acid-precipitable.Abbreviations: B lymphocyte, bone marrow-derived, thymusindependent lymphocyte; T lymphocyte, thymus-dependent lymphocyte.2879