Lymphocyte Cell Surface Molecules Research Articles

Distribution of central and effector memory T cells subsets in tumor infiltrating lymphocytes of HCC patients

Objective To illustrate the distribution of two memory T cells subsets CD45RO+ CD62L+ CCR7+ T cells (central memory T cells, TCM) and CD45RO+ CD62L-CCR7-T cells (effector memory T cells, TEM) in tumor infiltrating lymphocytes (TILs) of hepatocellular carcinoma (HCC) patients. Methods Polychromatic flow cytometry (PFC) was used to test the cell surface molecules of infiltrating lymphocytes in liver, including CD3, CD4, CD8, CD45RO, CD62L and CCR7. Results The proportions of CD4+ TCM subsets in CD4+ CD45RO+ Tm in HCC tissues, adjacent liver tissues and normal liver tissues were (0.25±0.17)%, (0.29±0.22)% and (0.37±0.33)%, and there were no significant differences (P>0.05). The proportions of CD4+ TEM subsets in CD4+ CD45RO+ Tm in HCC tissues, adjacent liver tissues and normal liver tissues were (89.7±7.4)%, (98.7±0.6)% and (95.5±3.7)%, respectively. Paired comparison showed that the proportions of CD4+ TEM subsets in HCC tissues were significantly lower than those in adjacent liver tissues and normal liver tissues (P<0.05). The proportions of CD8+ TCM subsets in CD8+ CD45RO+ Tm in HCC tissues, adjacent liver tissues and normal liver tissues were (0.41±0.31)%, (0.55±0.60)% and (0.26±0.25)%. Paired comparison showed that the proportions of CD8+ TCM subsets in adjacent liver tissues were significantly higher than those in normal liver tissues (P<0.05). The proportions of CD8+ TEM subsets in CD8+ CD45RO+ Tm from HCC tissues, adjacent liver tissues and normal liver tissues were (92.4±4.3)%, (98.1±0.7)% and (98.2±1.1)%. Paired comparison showed that the proportions of CD8+ TEM subsets in HCC tissues were significantly lower than those in adjacent liver tissues and in normal tissues (P<0.05). Conclusions Comparing to the TCM,TEM subsets including CD4+ and CD8+ , accounted for the majority of tumor infiltrating CD45RO+ Tm in HCC patients. The proportions of CD4+ TEM and CD8+ TEM subsets in CD45RO+ Tm from tumor tissues were significantly lower than those in non-cancerous tissues. However, the proportions of CD4+ TCM and CD8+ TCM subsets in CD45RO+ Tm from tumor tissues were the same as those from non-cancerous tissues. Key words: Central memory T cells; Effector memory T cells; Tumor infiltrating lymphocytes; Hepatocellular carcinoma

Antiphase signalling in the neuroendocrine-immune system in healthy humans

Background Any quantity varying in the spatial-temporal dimension may be considered as a signal. Human lymphocyte cell surface molecules and subsets present circadian variation and this variation may represent a kind of signalling in the neuroendocrine-immune system. We have analyzed the dynamics of variation of specific lymphocyte subsets in healthy humans. Subjects and methods In our study, lymphocyte subpopulation analyses were performed and cortisol, melatonin, GH and TSH serum levels were measured on blood samples collected every 4 h for 24 hours from eleven healthy men, ages 35–53 years (mean = 44 ± 6 SD). Results A clear circadian rhythm was validated for CD8 and cortisol with acrophase during the day and for CD3, CD4, melatonin, GH and TSH with acrophase at night. Cross-correlation showed that CD3 correlated positively with CD4 ( ρ = 0.67, P < 0.05) and negatively with CD8 ( ρ = −0.41, P < 0.05), CD4 correlated positively with melatonin ( ρ = 0.90, P < 0.05), GH ( ρ = 0.92, P < 0.05) and TSH ( ρ = 0.71, P < 0.05), negatively with CD8 ( ρ = −0.90, P < 0.05) and cortisol ( ρ = −0.18, P < 0.05), CD8 correlated positively with cortisol ( ρ = 0.38, P < 0.05). Discussion The different profiles of nyctohemeral changes of lymphocyte cell surface molecules and specific lymphocyte subsets realize different relationships with the neuroendocrine hormones and might represent a way of signal transmission among the multiple components of the neuroendocrine-immune system.

Polyreactive Monoclonal Antibodies Synthesized by Some B-CLL Cells Recognize Specific Antigens on Viable and Apoptotic T Cells.

B-cell chronic lymphocytic leukemia (CLL) is a disease characterized by uncontrolled clonal expansion of a CD5+/CD19+ B cell. Clones from different patients often express receptors/mAbs with remarkably similar VHDJH rearrangements, suggesting that the same antigen(s) or epitope(s) led to the initial clonal expansion. Although the specific antigens and epitopes remain to be identified, recent data indicate that B-CLL mAbs often bind autoantigens. In certain systemic autoimmune diseases, autoantibodies can react with cell surface molecules of viable lymphocytes (anti-lymphocyte mAbs) and with intracellular molecules of apoptotic cells that are transported to the cell membrane and function as autoantigens. Therefore, we expressed recombinant antibodies from B-CLL cells as IgG1 mAbs and tested them by flow cytometry for immunoreactivity with surface molecules on irradiated (apoptotic) and non-irradiated Jurkat cells, a human T-cell line. We chose to use T cells as our target for analysis to avoid difficulties that might arise from binding to FcgRIIb on B lymphocytes.Initial screening indicated that 5 of 25 mAbs showed intense binding. Antibodies from CLL Nos. 141 and DO8 bound to live but not apoptotic cells; in contrast, CLL Nos. 014, 114, and DO13 bound to apoptotic but not live cells. These five antibodies express unmutated VH genes. CLL 141 and DO8 express VH4-34. CLL 014 expresses VH1-69, CLL 114 VH4-39 and DO13 VH1-02. Other antibodies expressing VH1-69 in its unmutated form also bound Jurkat cells, albeit weaker than CLL 014, whereas mAbs expressing different unmutated genes from the VH1 family did not react. Among the mAbs expressing VH1-02 that were tested, only DO13 showed reactivity. This indicates that the VH1-02 gene was not solely responsible for the observed binding, and that DJ and/or VLJL segments were also involved. Remarkably, none of the tested mAbs belonging to VH3 family recognize antigens on Jurkat cells; all of these VH3 mAbs were mutated. Only CLL 114 belongs to a previously identified stereotypic set. Of interest, although CLL 014 and 141 are polyreactive, binding ssDNA, dsDNA, insulin, LPS, and HEp-2 cell lysates, their reactivities with surface antigens were specific in that CLL 141 bound only live cells and CLL 014 reacted solely with apoptotic cells.Our results indicate that, despite their inherent polyreactivity, certain B-CLL mAb bind specifically to antigenic epitopes on the surface of live as well as apoptotic Jurkat cells. The identity of these autoantigens is currently being investigated. These data highlight the importance of autoreactivity in the promotion and evolution of B-CLL.

Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.

Characterization of Prostate Cell Types by CD Cell Surface Molecules

A set of monoclonal antibodies raised against lymphocyte cell surface molecules, the cluster designation (CD) antigens, was used to distinguish the constituent cell types of the prostate. The luminal secretory epithelial, basal epithelial, fibromuscular stromal, nerve sheath, and endothelial cells express distinctive complements of cell surface molecules that were identified by immunohistochemistry using 152 commercially available antibodies. Many of the CD antibodies stained lymphocyte populations in the prostate. These lymphocyte populations were grouped into abundance classes of rare, moderate, and high. Some of these molecules are expressed by multiple cell types, both parenchymal and lymphoid; others are expressed by only one cell type. Distinctive patterns of CD expression, which are most similar to the expression pattern of prostate luminal cells, also characterize a small series of Gleason score 6 prostate cancers. The cell-type specificity of CD molecules increases the prospect of isolating specific cell populations, using such techniques as laser capture microdissection and flow cytometry, for cell-specific molecular studies.

Modulation of B Lymphocyte Antigen Receptor Signal Transduction by a CD19/CD22 Regulatory Loop

CD19 and CD22 are B lymphocyte cell-surface molecules that positively and negatively regulate antigen receptor signal transduction, respectively. Biochemical studies with B cells from CD19-deficient and CD22-deficient mice indicated that these two regulatory molecules influenced each other's functions: CD22 expression negatively regulated CD19 tyrosine phosphorylation, while optimal CD22 function was dependent on CD19 expression. Functional CD19 and CD22 interactions were also assessed in vivo by generating CD19/CD22 double-deficient mice. Remarkably, the CD19 mutation was dominant to the CD22 mutation in most instances. B lymphocytes from CD19/CD22-deficient and CD19-deficient mice were functionally equivalent despite the negative influence normally provided by CD22 expression. These data collectively suggest that CD19 activates the CD22/SHP1 inhibitory pathway that then acts primarily on CD19.

Jacalin, a lectin with anti-HIV-1 properties, and HIV-1 gp120 envelope protein interact with distinct regions of the CD4 molecule

Jacalin is a multimeric plant lectin able to interact with the lymphocyte cell-surface molecule CD4, a known receptor for the human immunodeficiency virus type 1 (HIV-1). Moreover, jacalin is able to block HIV-1 infection of CD4 + lymphoblastoid cells. Here we studied whether jacalin prevents HIV-1 gp120-CD4 interactions. We found (i) that jacalin did not inhibit HIV-1 Lai-induced syncytium formation that requires gp120-CD4 interactions; (ii) that jacalin prevented neither rgp120 binding to cell-surface CD4 nor sCD4 binding to viral envelope proteins expressed at the surface of HI V-1-infected lymphoblastoid cells; (iii) that jacalin did not compete for binding to CD4 with anti-CD4 mAb specific for the CDR2- or CDR3-like regions of the D1 domain of CD4; (iv) that jacalin did not bind a recombinant soluble molecule containing the D1/D2 domains of CD4; and, (iv) that jacalin binding to CD4 is inhibited by sugars known to interact with the lectinic-site of jacalin. These data have implications for the understanding of the mechanism by which jacalin blocks HIV-1 infection of CD4 + cells.

The Anti-CD6 mAb, IOR-T1, Defined a New Epitope on the Human CD6 Molecule That Induces Greater Responsiveness in T Cell Receptor/CD3-Mediated T Cell Proliferation

The T lymphocyte cell surface molecule, CD6, has been shown in a number of studies to play an important role in T cell activation. Its physiological ligand or function is still unknown. A panel of five anti-CD6 mAbs was used in the present study to investigate the structure-function relationship of this molecule. Cross-blocking assays indicate that three different epitopes were defined on the CD6 molecule by these mAbs. One of these epitopes defined by the mAb, IOR-T1, is insensitive to thiol-reducing agents, such as dithiothreitol and 2-mercaptoethanol. Of the other two epitopes, one was defined by 2H1 and the other was shared by three other mAbs, T12, 6D3, and Dako-CD6. All the CD6 mAbs at optimal concentration exhibit equal potency in enhancing T cell proliferation mediated through the T cell receptor/CD3 complex by optimal concentration of the anti-CD3 mAb, OKT3 (100 ng/ml). Simultaneous cross-linking of both the anti-CD6 mAbs and OKT3 is essential for the synergistic effect. When suboptimal concentrations of OKT3 (1 ng/ml) were used (no detectable cell proliferation), the synergistic effect of the anti-CD6 mAbs was still evident but with a differential effect. The epitope defined by IOR-TI consistently induced greater T cell responsiveness under these conditions. Our results suggest that the CD6 molecule may play an important role in T cell activation, and that signals through an epitope of stable conformation appear to be of importance when antigen levels are low or interacting with low-avidity antigen receptors.

標的リポソームを用いての薬剤の細胞内輸送に関する研究

Several parameters have been established as important for the efficient delivery of liposomes-associated molecules. A non-exhaustive list includes parameters, such as size of liposomes, lipid composition, nature of the encapsulated molecule, the nature of the target molecule and the cell type expressing it. Of these, we studied size of liposomes and target molecules on human T lymphocytes. Liposomes, composed of dipalmitoylphosphatidylcholin, cholesterol and the modified phosphatidylethanolamine used to couple Protein A, were used as a model system to demonstrate drug transfer into specific lymphoid cells. Protein A, which selectively recognizes mouse IgG2 antibodies, was coupled to liposomes to target them specifically to defined cells types coated with IgG2 antibody. Methotrexate(MTX)-containing liposomes of different sizes (0.05, 0.1, or 0.2 μm), targeted to the major histocompatibility complex(MHC)-encoded class I molecules, were evaluated by their efficiency. It was clearly demonstrated that large liposomes are internalized by human lymphoid cells less well than smaller liposomes. And we have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific drug delivery of drugs from targeted liposomes. CD? was shown to he a good target for drug delivery. But CD4 targeted liposomes, in contrast, was ineffective.

Generation and differentiation potential of retrovirus-immortalized mature murine B cells.

Given the availability of recombinant lymphokines, antibodies specific for various lymphokines and lymphocyte cell-surface molecules, and functional antigen-specific Th clones, homogeneous and functionally responsive B cell lines and clones would be valuable tools for studying T cell-B cell interactions and B lymphocyte differentiation. However, most of the available B cell lines to date have limited potential for differentiation. Generation of functional B cell lines has become an urgent task.

CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes

We have examined two T lymphocyte cell surface molecules, CD4 and CD7, as targets for specific delivery of drugs from antibody-directed liposomes. The efficiency of uptake by peripheral lymphocytes, thymocytes, and two CEM sublines (CEM.MRS and CEM-T4) of anti-CD4 and anti-CD7 liposomes containing methotrexate was evaluated by the methotrexate-mediated inhibition of the incorporation ofd-[3H]Urd into DNA. This was compared with similar liposomes targeted to MHC-encoded HLA class I molecules, which are known to be efficiently taken up by T cells. Despite the lower expression of CD7 molecules relative to HLA class I on most cell lines, CD7 was shown to be a good target for drug delivery. The results of an internalization study using radiolabeled Protein A showed that a higher proportion of CD7 molecules was internalized than HLA class I molecules. CD4-targeted liposomes, in contrast, were relatively ineffective for drug delivery for lymphoid cells, and only partially inhibited CEM-T4 cells. The lack of toxicity correlated with poor internalization of the target molecule on most cell lines. The drug effect of anti-CD4 liposomes was more pronounced on HeLa-T4, which is an epithelial cell line transfected with the CD4 gene. In contrast to lymphoid cells, these cells efficiently internalized CD4 molecules. PMA is known to down-regulate surface expression of CD4 molecules on various T cells. Internalization of CD4 was induced by PMA, but PMA failed to induce cytotoxicity of CD4-targeted liposomes for CEM.MRS. The internalized drug was probably degraded rapidly because internalized anti-CD4 antibody-bound Protein A was degraded very rapidly.

The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells.

Activation via the T lymphocyte cell surface molecule CD28 provides a potent amplification signal for interleukin 2 (IL-2) production in several in vitro systems. The B lymphocyte activation antigen, B7/BB1, is a natural ligand for CD28. Here we investigate the role of CD28 and B7/BB1 in primary activation of CD4+ T lymphocytes stimulated with allogeneic B lymphoblastoid cell lines. A subset of peripheral CD4+ T cells that is unresponsive to crosslinking of CD3/T cell receptor (TCR) with CD3 monoclonal antibody (mAb) does proliferate in response to allogeneic B lymphoblasts. TCR binding to allogeneic major histocompatibility complex antigens was an absolute requirement for activation of these cells because mAbs to either CD3 or human histocompatibility leukocyte antigen (HLA) class II completely inhibited activation. CD28 and B7/BB1 antibodies inhibited T cell proliferation 90% and 84%, respectively. Similar results were obtained with the total CD4+ T lymphocyte population. Crosslinking of HLA-DR antigens on small, resting B cells induced rapid expression of B7/BB1, which peaked at 6 h and returned to baseline levels within 18 h. These data demonstrate that CD28-B7/BB1 binding provides an important early second signal for alloactivation of CD4+ T lymphocyte by B lymphoblasts. The results also suggest that T cells interacting with allogeneic resting B cells may induce B7/BB1 expression in the alloantigen-presenting cell as a consequence of interaction between the TCR and class II molecules.

The polyclonal activation of lymphocytes and T cell mitogenicity by a unique sialic-acid-binding lectin from the hemolymph of Achatina fulica snail

A unique sialic-acid-binding lectin, AchatininH, isolated from the hemolymph of Achatina fulica snail was found to be strongly mitogenic, as monitored by [3H]thymidine incorporation assay, to rat and human lymphocytes. However, the degree of mitogenic response varied with the type of cell population. As indicated, this lectin induced proliferation of purified T lymphocytes and rat thymocytes, whereas it was less mitogenic towards peripheral lymphocytes of pregnant rats and was not mitogenic towards B lymphocytes. Furthermore, the mitogenic response was inhibited by the sialic-acid-containing disaccharide, a strong inhibitor of this lectin. This suggests that lymphocyte cell surface molecules containing sialic acid residues are important for this interaction and may have a structure similar to that of AchatininH receptors. Although this lectin showed strong mitogenicity towards lymphocytes, it showed very weak leucoagglutination. Surprisingly, PHA-induced blastogenesis was inhibited by the same dose of AchatininH as caused mitogenic activity in resting lymphocyte culture. The degree of suppression was higher for the lymphocytes isolated from pregnant rat blood compared to the non-pregnant control. Since the 51Cr-uptake assay and the cell viability test results negate the potential cytotoxic activity of this protein, the immunosuppression induced in the presence of PHA by AchatininH may be the additive effect of two mitogens causing an increased cell-density-dependent arrest. AchatininH does not have complement-like activity, but induces potent polyclonal activation of B cells as measured by the reverse hemolytic plaque assay.

Immunophenotypic and cell cycle analysis of lymph node cells from dimethylbenzanthracene-treated mice

The chemical carcinogen 7, 12-dimethylbenz-(a)anthracene (DMBA) depletes Langerhans cells from murine epidermis. Application of contact sensitizers to DMBA-treated skin induces specific immunological tolerance due to a DMBA-resistant epidermal antigen presenting cell (APC) migrating to local lymph nodes where it presents antigen in a way which activates suppressor cells. As alterations in local lymph node lymphocytes may enhance the ability of the DMBA-resistant APC to activate suppressor cells, these cells were examined in DMBA-treated mice. Lymph nodes in DMBA-treated mice had normal morphology but were larger and contained increased numbers of lymphocytes. Cell cycle analysis revealed that these lymphocytes did not arise from division within the lymph node, suggesting alterations in homing properties of lymphocytes. Contact sensitizer applied to DMBA-treated skin did not increase lymphocyte division, possibly due to suppressor cell inhibition of the development of effector lymphocytes. DMBA treatment had no effect on B cells or Ia expression, but decreased levels of the T lymphocyte cell surface molecule Thy-1, and increased L3T4 and Lyt-2 as quantitated by flow cytofluorimetry. These changes could influence the development of immune responses as these T cell molecules are receptors involved in lymphocyte interactions.

Isolation and chromosomal localization of cDNAs encoding a novel human lymphocyte cell surface molecule, LAM-1. Homology with the mouse lymphocyte homing receptor and other human adhesion proteins.

A cDNA encoding a new human lymphocyte cell surface molecule has been isolated and shown to identify a fourth member of a recently discovered family of adhesion proteins. This lymphocyte-associated molecule (LAM-1) is uniquely composed of multiple distinct domains, one domain homologous with animal lectins, one homologous with epidermal growth factor, and two short consensus repeat units similar to those found in C3/C4 binding proteins. This cDNA clone hybridized with RNAs found in B cell lines and T lymphocytes, but not with RNA from other cell types. The amino acid sequence of LAM-1 is 77% homologous with the sequence of the mouse lymphocyte homing receptor, suggesting that LAM-1 may function in human lymphocyte adhesion. The LAM-1 gene is located on chromosome 1q23-25, as is another member of this adhesion family, suggesting that this new family of proteins may be encoded by a cluster of "adhesion protein" loci.

A large proportion of T lymphocytes lack CD5 expression after bone marrow transplantation

The lymphocyte cell surface molecule CD5 (T1, Leu 1, Tp67 in the human; Ly 1 in the mouse) is expressed on the majority of circulating T lymphocytes and a small population of B cells. We have analyzed CD5 expression on repopulating T cells in the peripheral blood of patients after allogeneic bone marrow transplantation (BMT). The frequency of CD3+ T cells that lack expression of CD5 is dramatically increased after BMT compared with the normal population. The percent of total CD3+ CD5- cells correlated with the presence of graft versus host disease and with time following transplant, but did not correlate with age, diagnosis, preparative regimen, T-cell depletion of the marrow, major histocompatibility complex compatibility, or the presence or absence of interstitial pneumonitis. Furthermore, the total number and percent of CD8+ CD5- cells was increased following BMT. CD3+ cells from BMT patients were sorted for the presence or absence of CD5 expression. CD3+ CD5- cells were capable of interleukin-2 production and of mediating cytolysis following lectin stimulation. We conclude that CD3+ CD5- T cells are functional and represent a significant proportion of circulating cells in patients after BMT.

A Large Proportion of T Lymphocytes Lack CD5 Expression After Bone Marrow Transplantation

Abstract The lymphocyte cell surface molecule CD5 (T1, Leu 1, Tp67 in the human; Ly 1 in the mouse) is expressed on the majority of circulating T lymphocytes and a small population of B cells. We have analyzed CD5 expression on repopulating T cells in the peripheral blood of patients after allogeneic bone marrow transplantation (BMT). The frequency of CD3+ T cells that lack expression of CD5 is dramatically increased after BMT compared with the normal population. The percent of total CD3+ CD5- cells correlated with the presence of graft versus host disease and with time following transplant, but did not correlate with age, diagnosis, preparative regimen, T-cell depletion of the marrow, major histocompatibility complex compatibility, or the presence or absence of interstitial pneumonitis. Furthermore, the total number and percent of CD8+ CD5- cells was increased following BMT. CD3+ cells from BMT patients were sorted for the presence or absence of CD5 expression. CD3+ CD5- cells were capable of interleukin-2 production and of mediating cytolysis following lectin stimulation. We conclude that CD3+ CD5- T cells are functional and represent a significant proportion of circulating cells in patients after BMT.

A secreted form of the human lymphocyte cell surface molecule CD8 arises from alternative splicing.

The human lymphocyte differentiation antigen CD8 is encoded by a single gene that gives rise to a 33- to 34-kDa glycoprotein expressed on the cell surface as a dimer and in higher molecular mass forms. We demonstrate that the mRNA is alternatively spliced so that an exon encoding a transmembrane domain is deleted. This gives rise to a 30-kDa molecule that is secreted and exists primarily as a monomer. mRNA corresponding to both forms is present in peripheral blood lymphocytes, Con A-activated peripheral blood lymphocytes, and three CD8+ T-cell lines, with the membrane form being the major species. However, differences in the ratio of mRNA for membrane CD8 and secreted CD8 exist. In addition, the splicing pattern we observe differs from the pattern found for the mouse CD8 gene. This mRNA is also alternatively spliced, but an exon encoding a cytoplasmic region is deleted, giving rise to a cell surface molecule that differs in its cytoplasmic tail from the protein encoded by the longer mRNA. Neither protein is secreted. This is one of the first examples of a different splicing pattern between two homologous mouse and human genes giving rise to very different proteins. This represents one mechanism of generating diversity during speciation.

Structural studies of T200 glycoprotein and the IL-2 receptor.

Immunological analysis of the cell surface of hematopoietic cells has led to the identification of many different cell membrane molecules, some of which have well-defined functions as receptors. In general, however, the role of most lymphocyte cell surface molecules remains ill-defined even in cases in which antibody inhibition studies have given some insight into the biological processes in which they participate. Here we describe molecular and biochemical studies of T200 glycoprotein (leukocyte-common antigen) and the IL-2 receptor which illustrate the kinds of approaches that can be currently used to characterize individual molecules. T200 glycoprotein is a large Mr glycoprotein found exclusively on leukocytes. However, the exact Mr varies in a cell-type-specific fashion and this property is conserved between different species. Comparison of the rat, mouse and human cDNA sequences show that the large cytoplasmic portion of the molecule is well-conserved, approximately 90%, whereas the exterior portion is only about 50% homologous. Cell-type-specific differences in the primary sequence of the molecule have been identified in the N-terminal portion of the molecules. In contrast to T200, the function of the IL-2 receptor is well-known. The interaction of IL-2 with its receptor provides a growth signal that determines the magnitude and duration of T-cell responses. Limited proteolysis studies provide the first direct biochemical evidence that the external region of the IL-2 receptor consists of two independent domains. 125I-labeled IL-2 has been chemically crosslinked to the receptor and proteolytic cleavage of the crosslinked product indicates that IL-2 is selectively bound to the N-terminal domain of the receptor.

Fractionation of detergent lysates of cells by ammonium sulphate-induced phase separation

A procedure is described for fractionating detergent lysates of cells based on the ability of (NH 4) 2SO 4 to induce phase separation of detergents such as Triton X-100, sodium deoxycholate, and sodium cholate, into detergent-rich and detergent-depleted phases. An analysis of six murine lymphocyte cell surface molecules revealed that the partitioning in Triton X-100 of each molecule was highly dependent upon the (NH 4) 2SO 4 concentration, each antigen partitioning into the detergent-rich phase at a defined salt concentration. In contrast, none of the six molecules appeared in the detergent-rich phase of a Triton X-114 phase separation, even though two of the molecules, namely Ly- 2 3 and L3T4, are well-characterized integral membrane proteins. It was also observed that (NH 4) 2SO 4 resulted in the partitioning of many nonmembrane proteins into the detergent-rich phase, indicating that the procedure can be used to fractionate all cellular proteins. By judicious choice of (NH 4) 2SO 4 concentrations, precipitation of cellular proteins at two different (NH 4) 2SO 4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.