Human migration inhibitory factor: purification and immunochemical characterization.

Using gel filtration and preparative isotachophoresis, the migration inhibitory factor (MIF) was highly purified from human lymphocytes activated with concanavalin A. MIF is an acidic protein with a mol wt of approximately equal to 25,000 daltons as determined by gel filtration and analytical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The protein inhibits migration of macrophages in the capillary test and in addition, has a slowing effect on the electrophoretic mobility of guinea pig peritoneal macrophages. Rabbit antibodies specific for this protein, as determined by immunochemical techniques, neutralized the biological effect of MIF on migration and on the electrophoretic mobility of macrophages.

Immunoglobulins on the surface of lymphocytes. I. Distribution and quantitation.

The distribution, and quantity of immunoglobulins on the surface of lymphocytes has been studied by means of immunofluorescence and a quantitative radio-immunoassay. Surface immunoglobulins were found on approximately 45% of spleen and marrow lymphocytes and 7-14% of lymphocytes from lymph nodes, peripheral blood, and peritoneal exudate. Thymic lymphocytes contained undetectable amounts of immunoglobulin. In the spleen the different immunoglobulins were present in the following order: gammaG2 > gammaG1 > M > gammaA > gammaG3. The surface immunoglobulin was largely removable by brief treatment with trypsin. Quantitative analysis indicated that 50,000-150,000 molecules of immunoglobulin were present on an individual cell. A variety of observations make it likely that this lymphocyte-associated immunoglobulin. is a product of the cell to which it is attached rather than a form of cytophilic antibody.

Human T cell receptor V beta gene polymorphism.

Southern blot hybridizations with human T cell receptor V beta gene probes were used to determine the sizes of the various V beta gene subfamilies. An analysis of DNA samples from 100 unrelated individuals identified a single individual who lacked one V beta gene segment. A second individual had an apparently different repertoire of V beta gene segments in one subfamily, as assayed by hybridization, possibly due to a gene conversion event. An analysis with four restriction enzymes of DNA from 30 consanguineous donors detected restriction fragment length polymorphisms associated with 12 of 14 V beta gene segment subfamilies examined. In an analysis of DNAs from a large panel of unrelated individuals, some alleles at these loci were found to be in linkage disequilibrium, indicating a potentially close physical linkage. The segregation of three polymorphisms, two associated with V beta gene segment loci and one associated with the C beta genes, was compatible with Mendelian inheritance, and demonstrated that highly informative haplotypes could be generated. The high degree of polymorphism observed in the human T cell receptor beta chain complex should allow exploration of possible associations between T cell receptor genes and inherited diseases involving the immune system.

A novel subset of human lymphocytes with a T cell receptor-gamma complex.

We have previously characterized a CD3+ T cell receptor (TCR) alpha/beta- human fetal cloned cell line, termed F6C7, which surface-expresses a CD3-associated gamma chain identified by anti-NKFi, an mAb with a restricted clonotypic reactivity. Here, we have produced an additional antibody, anti-Ti-gamma A, which recognizes a public epitope of the gamma molecule defined by anti-NKFi. Ti-gamma A is present on approximately 3% of circulating lymphocytes with a wide range (1-15%) among 30 healthy individuals tested. Two-color immunofluorescence experiments performed with anti-Ti-gamma A and BMA 031 mAb (a reagent specific for the TCR-alpha/beta receptor) showed that surface expression of Ti-alpha/beta and Ti-gamma A is mutually exclusive. Moreover, it was found that most Ti-gamma A+ cells are CD2+, CD3+, CD4-, CD5+, NKH1-, HLA class II-negative. In contrast, the expression of the CD8 molecule on these T lymphocytes appears to be variable from one individual to another. Finally, we found that Ti-gamma A+ cells represent a majority of peripheral lymphocytes that express CD3 proteins but not the TCR-alpha/beta heterodimer. The delineation of this unique lymphocyte subset should help further studies on the biology of cells with a CD3-associated gamma complex.

Some physical and radiobiological properties of immunologically reactive mouse spleen cells.

Three classes of immunologically reactive cells, differing only slightly in size from each other, are required for the production of hemolysin-forming cells in culture. The three classes of cells can be detected in the normal mouse spleen by the combined use of rosette formation, velocity sedimentation, and irradiation. One class of cells (peak sedimentation velocity, 3.2 mm per hr) forms rosettes. The capacity of these cells to participate in immune responses to foreign erythrocytes is inhibited by relatively low doses of irradiation. These cells may be the immediate precursors of hemolysin-forming cells. A second class of cells (peak sedimentation velocity, 3.6 mm per hr) facilitates the production of hemolysin-forming cells by small numbers of normal spleen cells. Their facilitative activity is resistant to a relatively large dose of radiation. They do not form rosettes. The requirement of a third class of cells was deduced from the results of mixing experiments. Neither rosette-forming cells nor spleen cells largely depleted of rosette-forming cells could give rise to hemolysin-forming cells when cultured either alone or in the presence of large numbers of heavily irradiated cells. However, when rosette-forming cells, cells depleted of rosette-forming cells, and heavily irradiated cells were mixed together, hemolysin-forming cells were produced. The peak responses were found in fractions sedimenting at 4 mm per hr. Thus, it is suggested that these fractions contain a third class of cells. This class of cells does not form rosettes, but its function is inhibited by relatively low doses of radiation.

Characterization of DNA excreted from phytohemagglutinin-stimulated lymphocytes.

The DNA released into the culture medium after phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes has been purified and characterized. It is double stranded, sediments at 7-8S in alkaline sucrose, and has a Tm determined optically and by thermal elution from hydroxyapatite that is substantially lower than that found for lymphocyte cell DNA. Media DNA contains a major component reassociating with an average Cot-1/2 of 87 mol X s/liter, compared to a Cot-1/2 of 770 mol X s/liter for the unique fraction of cell DNA as measured by reassociation in 0.6 M Na+. This component of media DNA consists of unique sequence elements which are largely shared in media DNA preparations from cultures derived from different cell donors. The marked difference between media DNA and cell DNA indicates that media DNA is not derived from cell death and lysis, rather than some unique portion of lymphocyte DNA is apparently excreted from the cells during PHA-stimulated growth.

Restriction fragment length polymorphism of the gene encoding the alpha chain of the human T cell receptor.

Two allelic forms of the T cell antigen receptor alpha chain gene were discerned by restriction fragment length polymorphism (RFLP) employing the T cell antigen receptor alpha chain probe pGA5, and the restriction enzyme Bgl II. Analysis revealed that the polymorphic fragments are detected by a probe specific for the constant region exon of the T cell antigen receptor alpha chain gene. Furthermore, the polymorphic fragments were shown to segregate within families. The two allelic forms yield two homozygous states, 3.2/3.2 and 2.9/2.9, at a frequency of 76.5 and 2.9%, respectively, within the normal population. The heterozygous state was observed in 20.6% of the population. The discovery of allelic forms of both the alpha and beta chains of the T cell antigen receptor genes may provide a unique opportunity to study heritable markers of T cell function in several human diseases.

Human macromolecular insoluble cold globulin (MICG). I. T-cell origin of T-MICG and null cell origin of N-MICG.

Although surface immunoglobulin characterizes B cells in man, there are few surface markers that distinguish T cells. We have described a new protein synthesized in human T cells, termed T-MICG. This protein is a macromolecule of 225,000 daltons, is insoluble in the cold, and migrates as a beta-globulin on electrophoresis. Separation of human peripheral blood lymphocytes into T and B-cell populations by rosette sedimentation and anti-human-Fab columns clearly demonstrated the T-cell origin of the 225,000 dalton component. Furthermore, null cells were shown to synthesize a protein of 185,000 daltons, termed N-MICG, with physical properties similar to T-MICG, T-MICG and N-MICG were shown to be antigenically dissimilar, employing antiserum to each of these proteins. The present studies demonstrate two novel cell surface markers, T-MICG and N-MICG, which characterize T cells and null cells, respectively.

Characterization of HLA-D-region antigens by two-dimensional gel electrophoresis. Molecular-genotyping.

Monoclonal antibodies directed against nonpolymorphic determinants of HLA-D/DR molecular complex (human Ia antigens) were used to immunoprecipitate HLA-D-region-associated molecules from [35S]methionine internally labeled and 125I surface-labeled B lymphoblastoid B cell lines. Analysis of these by two-dimensional nonequilibrium-pH-gradient electrophoresis reveals a molecular heterogeneity within a 26,000- to 34,000-mol wt range. At least three sets of spots are identified: a very acidic set of 32,000- to 34,000-mol wt, a very intense invariant spot of 31,000 mol wt, and a series of 26,000- to 29,000-mol wt spots of variable charge. Comparison of immunoprecipitates from nine different HTC demonstrates that the polymorphism is localized in this latter set. Pulse-labeling and inhibition of glycosylation by tunicamycin show that the electrophoretic polymorphism is of polypeptide origin, whereas N-linked oligosaccharrides and sialic acid residues contribute to the microheterogeneity of the profile. Two-dimensional gels provide an electrophoretic genotyping procedure and allow analysis of the genetic organization and molecular complexity of the HLA-D/DR molecules.

Studies of the cells in the afferent and efferent lymph of lymph nodes draining the site of skin homografts.

Intermediate lymph (efferent from the prefemoral lymph node) was collected for 600 hr from both flanks of each of four sheep that had an autograft of skin on the left flank and a homograft of skin on the right flank. 8 days after the grafts had been applied considerable numbers of large basophilic cells, apparently identical with those that appear during immune responses to conventional antigens, appeared in the lymph draining from the homografts. No such cells appeared in the lymph draining from the autografts. At this time the homografts were already showing signs of rejection and were apparently dead well before the cellular response in the lymph reached a peak, about 350 hr (14-15 days) after the homografts had been applied. During the peak of the response up to 40% of the cells in the lymph were basophilic cells and in one experiment such cells were leaving the lymph node at a rate of 200 million per hr. Peripheral lymph (afferent to the popliteal lymph node) draining from the sites of homografts of skin was collected from five sheep. This lymph contained few white cells (<1000 per mm(3)) and showed only an insignificant lymphoid cell reaction. Although the percentage of macrophage-like cells was increased significantly there were few signs of a lymphoid cell reaction; the lymph also contained much amorphous debris. Experiments in which the basophilic cells from the efferent lymph were labeled in vitro with thymidine-(3)H and returned to the sheep by intravenous injections were carried out in six sheep. The presence of the labeled cells in the grafts, blood, and other tissue was detected by liquid scintillation counting of nucleic acid extracts of biopsy and postmortem material and by radioautography. 2-3 labeled cells out of every 1000 injected entered the homografts but hardly any entered the autografts. However, labeled basophilic cells that had originated in response to bacterial antigens entered the homografts with equal facility. It is thus hard to believe that the immunological specificity of a lymphoid cell endows it with a specific "homing" capability. Furthermore, in all the experiments the specific radioactivities of the nucleic acids extracted from the blood mononuclear cells were approximately of the same order as those of the nucleic acids extracted from the homografts. It was concluded that most of the mononuclear cells that infiltrate homografts represent a random selection from the mononuclear cell population of the blood.

Antifluorescein affinity columns. Isolation and immunocompetence of lymphocytes that bind fluoresceinated antigens in vivo or in vitro.

A new method for the isolation of specific immunocompetent lymphocytes has been described in which lymphocyte populations are exposed to fluoresceinated antigens (FLAGs) in vivo or in vitro, and the FLAG-binding cells retained on antifluorescein affinity columns. Specific cells are then eluted with an unrelated FL-labeled protein and shown to be fully immunocompetent. This methodology has been applied successfully in diverse antigenic systems including polymerized flagellin and TNP-specific B cells and alloantigen-reactive cytotoxic T lymphocytes. The method is rapid, inexpensive (requiring only antifluorescein beads), and can be applied to any antigens (or antibodies) in which a fluorescein group can be introduced.

Relationships of gp70 of MuLV envelopes to gp70 components of mouse lymphocyte plasma membranes.

The family of glycoproteins called gp70 includes molecules that are the main constituent of murine C-type viral envelopes, and some that are expressed as mendelian constituents of thymocyte plasma membranes in the absence of virions. To investigate further the relation of viral gp70s to plasma- membrane gp70s we compared peptide maps of gp70s derived by immunoprecipitation from cells infected with chosen viruses and from various thymocytes and leukemiacells known to express one or more of three immunogenetically defined gp70 types: Glx-gp70, X-gp70, and O-gp70. Maps of gp70 from cultured cells infected with ecotropic and xenotropic viruses were distinguishable from one another, and in general resembled gp70 maps prepared directly from ecotropic and xenotropic virions respectively. Maps of gp70s immunoprecipitated from thymocytes of five mouse strains and from two A strain T-cell leukemias also fell into two distinguishable and generally corresponding patterns. Thus peptide-mapping substantiates earlier conclusions that viral gp70s and plasma-membrane gp70s inherited independently of virus-production are highly related or identical molecules. The gp70 maps of thymocytes from B6, B6-G(+IX), 129, and A mice formed a group resembling the map from cultured cells infected with xenotropic virus. Thymocytes from AKR mice, and the two A strain leukemias, gave gp70 maps conforming more to the second pattern, that of cultured cells infected with ecotropic virus. This second pattern probably comprises at least two gp70 types, one of which is X-gp70. Our data indicate that the G(IX)-gp70 and O-gp70 sub-species of gp70 expressed in the cell populations we have studied are coded by xenotropic viral genomes, and X-gp70 by ecotropic viral genomes.

Localization of immunological complexes fixing beta1C (C3) in germinal centers of lymph nodes.

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the beta(1C) component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit beta(1C), IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, beta(1C), and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for beta(1C), IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the beta(1C) component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.

The effect of neuraminidase on the fate of transfused lymphocytes.

Evidence has been obtained that incubation of rat lymphocytes with neuraminidase, prior to intravenous transfusion into allogeneic or syngeneic recipients, alters the distribution of the cells. Many enzyme-treated lymphocytes initially become trapped in the liver, and there is a decrease in the selective accumulation of these cells in the lymph nodes and spleen. Subsequently, many enzyme-altered cells emigrate from the liver, concentrate in lymph nodes, and recirculate to the lymph. The results suggest that sialic acid constituents of the lymphocyte surface play a critical role in ensuring the normal distribution of these cells in the body. The findings also imply that reactions involving surface sialic acid can markedly alter the fate of lymphocytes without "killing" the cells.

Cell surface immunoglobulin. XI. The appearance of an IgD-like molecule on murine lymphoid cells during ontogeny.

An Ig molecule containing L chains and H chains similar to human delta-chains has been detected on the surface of radioiodinated murine lymphoid cells. Newborn mice have only IgM on their splenocytes. Between 10 and 15 days, the IgD-like molecule appears and increases in amount until 3 mo of age, when it is the predominant cell surface Ig in terms of radioactivity. IgD is found only in peripheral lymphoid tissues and is present in larger amounts on peripheral lymph node cells (approximately 85% of surface Ig) than on splenocytes (approximately 50%). IgD is also present in comparable amounts on cells from both nu/nu and germfree mice, indicating that its expression may be independent of both thymic influence and antigenic stimulation. These studies suggest that there is a switch from cell surface IgM to IgD that occurs during differentiation of virgin B lymphocytes in the spleen.

The uropod-bearing lymphocyte of the guinea pig. Evidence for thymic origin.

In this study, the frequency of uropod formation and the type of lymphocyte bearing the uropod was investigated in various guinea pig lymphocyte populations. Without prior in vitro stimulation, almost 40% of peritoneal exudate lymphocytes (PELS) form uropods, while thymocytes and lymph node cells form far fewer. Subsequent stimulation in vitro with purified protein derivative demonstrated that there is an association between antigen reactivity and frequency of uropod formation in these populations. The ultrastructure of these uropods is identical to that described for human lymphocytes stimulated with phytohemagglutinin. In the populations studied, all the lymphocytes forming uropods lack easily detectable surface membrane immunoglobulin and are therefore most likely thymus-derived or T lymphocytes.

The influence of intracellular levels of cyclic nucleotides on cell proliferation and the induction of antibody synthesis.

The intracellular ratio of adenosine 3',5'-cyclic monophosphate (cyclic AMP) to guanosine 3',5'-cyclic monophosphate (cyclic GMP) may control the developmental pathway followed by antibody-forming cell (AFC) precursors. The evidence for this is derived from several different types of experiments. First lipopolysaccharide (LPS) which is mitogenic for B lymphocytes, stimulates rapid, transient changes in intracellular levels of cyclic GMP but not cyclic AMP when added to mouse spleen cultures. Cyclic GMP itself stimulates DNA synthesis in these cultures, suggesting that the intracellular changes in cyclic GMP levels are involved in the mitogenic signal delivered by LPS to cells. The absolute amounts of cyclic nucleotides may vary widely in different cells under various conditions, however, the intracellular ratio of cyclic AMP to cyclic GMP is always high in nondividing cells and low in dividing cells. AFC precursors appear to respond to antigen in the absence of T-cell activity by inactivation (1-7). In the response to antigen in the presence of specific T cells, precursor cells proliferate and mature to AFC. Raising intracellular levels of cyclic AMP inhibits cell proliferation and leads to precursor cell inactivation (14, 15). It is suggested that the interaction of antigen with immunoglobulin receptors on the surface of precursors cells leads to the stimulation of adenylate cyclase activity and initiates the inactivation pathway. Since cyclic GMP stimulates immune responses in T-cell-depleted cultures (14, 15) and increasing cyclic GMP levels appear to be involved in the delivery of a mitogenic signal to cells, it is suggested that T-helper cells deliver a signal to precursor cells via the stimulation of guanylate cyclase to initiate the inductive pathway. It is suggested that it is the intracellular ratio of cyclic AMP to cyclic GMP that regulates the fate of precursor cells, not the absolute level of one cyclic nucleotide.

Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes in situ.

Immunofluorescence analysis of mammalian lymphocytes using antiserum directed against chicken erythrocyte alpha-spectrin revealed a lymphocyte population in which spectrin antigen was arranged in the form of a discrete cap (hereafter referred to as capped lymphocytes). This subset could be easily distinguished from other lymphocytes in which the spectrin antigen was diffusely distributed near the plasma membrane (noncapped lymphocytes). The subset of capped lymphocytes could be visualized in situ and in isolated cells in the absence of added ligand. Using frozen sections of lymphoid organs that were fixed in formaldehyde prior to the immunofluorescence procedure, capped lymphocytes were found in characteristic locations depending on the tissue examined. In the thymus, the major population of medullary lymphocytes were capped whereas cortical lymphocytes were mostly noncapped. In Peyer's patches, capped lymphocytes were interspersed with noncapped lymphocytes throughout the tissue. In the spleen, capped lymphocytes were concentrated in the periarterial lymphoid sheath of the white pulp and in lymph nodes they were found predominantly in the paracortical and cortical regions. Capped lymphocytes were not visible in the thymus until just before birth and did not appear in the spleen until 3 d after birth. When lymphocytes were isolated from lymphoid organs, fixed in formaldehyde and prepared for immunofluorescence, capped and noncapped lymphocytes were still identifiable and present in the same relative proportions as seen in situ. Results identical to those described above are obtained using antisera directed against guinea pig fodrin. Natural capping of proteins previously shown to co-migrate with a variety of cell surface macromolecules after cross-linking may be a new means of identifying various stages of lymphocyte activation or differentiation.

Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes.

We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens. The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase. Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen. Thus, the proteins appear to be major components of activated cells. We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation. The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it. Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress. P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H. R. B. 1986. Cell. 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD). The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types. The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.

Lymphocyte alpha-actinin. Relationship to cell membrane and co-capping with surface receptors.

Mouse spleen lymphocytes synthesize a protein which comigrates with skeletal muscle alpha-actinin on two-dimensional gel electrophoresis and is immunoprecipitated by an antibody directed against skeletal muscle alpha-actinin. Mouse lymphocyte alpha-actinin is present in membrane fractions, and is immunoprecipitated from lymphocyte detergent lysates by an antiserum made against these purified membranes. The anti-alpha-actinin activity of this antiserum is not adsorbed after incubation with fixed intact lymphocytes. Lymphocyte alpha-actinin does not bind concanavalin A and it is inaccessible to lactoperoxidase-catalyzed surface iodination. Double immunofluorescence shows that alpha-actinin moves concurrently along the cell membrane with redistributed surface immunoglobulins and Thy-1 antigen, and remains associated up to 30 min with surface aggregates of these receptors. Our results suggest that lymphocyte alpha-actinin, as defined by molecular weight and cross reactivity with the antibody against the muscle protein, (a) is associated with the cell membrane, (b) is not expressed at the cell surface, and (c) participates in the movement of surface receptors.