Evidence that murine hematopoietic cell subset marker J11d is attached to a glycosyl-phosphatidylinositol membrane anchor

Glycosyl-phosphatidylinositol (G-PI) has been shown to serve as membrane anchor for cell surface molecules such as Thy-1, Ly-6-controlled ThB and Qa antigens. Here, we present several lines of evidence indicating that the hematopoietic cell lineage (i.e. thymocytes, B cell subset and red blood cells) marker defined by the rat monoclonal antibody J11d is also a G-PI-linked structure. First, surface expression of the J11d-defined molecules, and that of the related antigen B2A2, was found to be specifically reduced by treatment of thymocytes and B lymphoma or hybridoma cells with excess of Staphylococcus aureus PI-specific phospholipase C; this enzyme also solubilizes a 35-40-kDa material from erythrocyte microsomal membranes corresponding to the predominant J11d-reactive red cell surface molecules. Second, Thy-1- mutants of the BW5147, T1M1, S1A or S49 murine T lymphoma cells of the complementary classes A, B, C and E (i.e. shown to be defective in the enzymatic machinery that posttranslationally modify Thy-1 molecules) also lack J11d, or express it at a very low level. Although directed at a G-PI-linked structure, the J11d monoclonal antibody, unlike other reagents to Thy-1 or Ly-6-controlled antigens, failed to induce thymocyte proliferation even in the presence of phorbol myristate acetate and cross-linker monoclonal antibody.     

T cell activation via the CD2 molecule is associated with protein kinase C translocation from the cytosol to the plasma membrane

T cell activation via the CD2 molecule involves phospholipase C and phosphoinositide hydrolysis. Here we demonstrate that the triggering of subclones of the human T leukemia Jurkat cell line by anti-CD2 as well as anti-CD3 monoclonal antibodies is able to induce activation (i.e. translocation from cytosol to cell membrane) of protein kinase C (PKC), which is dependent on the formation of 1,2-diacylglycerol from inositol 4-5-bisphosphate. The kinetics of PKC translocation parallels the rise in intracellular calcium following both CD2 and CD3 stimulations. These results further demonstrate that CD2 and CD3 activation pathways use similar signal transduction mechanisms.     

Identification of a previously unrecognized polypeptide associated with lymphocyte function associated antigen one (LFA-1)

In lymphocyte function associated antigen one (LFA-1) preparations from metabolically labeled lymphocytes we have observed a new polypeptide component of 86-kilodalton additional to the already described - and β-chains. This chain is cosynthesized with the - and β-chains and can be covalently cross-linked with them, resulting in a three-chain complex. This complex is recognized by the H35–89.9 anti-LFA-1 monoclonal antibody. Cleveland peptide mapping analysis indicates that the new chain is structurally different from the - and β-chains of the LFA-1 complex. The chain has been observed in B-cells as well as in T-cells. Labeling properties of the 86-kilodalton chain suggest that this molecule is not exposed on the membrane.     

Two novel phospholipid-linked mouse thymocyte surface molecules released by phosphatidylinositol-specific phospholipase C

We searched for mouse thymocyte surface proteins attached to the cell membrane through a phosphatidylinositol (PI)-containing glycolipid similar to that identified in the T cell-activating Thy-1 glycoprotein. Our approach was to biochemically analyse the supernatants of ¹²⁵I surface-labeled thymocytes treated with 60 U/ml of Staphylococcus aureus PI-specific phospholipase C (PI-PLC). In addition to Thy-1, two molecules of M_r, 13,000 and 52,000 were found to be specifically solubilized by the enzymatic treatment. The 52,000 structure is a single basic polypeptide of M_r, 50,000 under non-reducing conditions. Two-dimensional gel electrophoresis analyses resolved the 13,000 mol. wt molecules in three relatively basic components including (i) a monomeric molecule(s), a fraction of which exhibited slower migration in reducing gels, and (ii) disulfide-linked multimeric structures comprising a major component of M_r, 30,000 and a minor one of M_r, 45,000. These 52,000 and 13,000 mol. wt molecules could be released from thymocytes and Escherichia coli lipopolysaccharide (LPS)-stimulated B cell blasts, but not from a variety of mature T cell populations. These data add new members to the list of PI-linked rodent lymphoid cell differentiation markers, which already includes three activation signal-transducing T cell molecules (i.e. Thy-1, Ly-6-linked T cell-activating proteins, and RT-6).     

T cell-mediated cytolysis: on the strength of effector-target cell interaction

Allosensitized lymphoid cell populations contain T lymphocytes that can bind to target cells and lyse them. We asked whether there was a relationship between lymphocyte target cell-binding strength and occurrence of cytolysis. Using graded shear forces to dissociate effector-target cell conjugates, we found that (a) within an allosensitized lymphoid cell population derived from an heterogeneous mixed leukocyte culture, there were lymphocyte-target cell conjugates with binding strengths differing by a factor of at least 10(2), (b) even the minimal force required to release a significant amount of bound target cells could disrupt the plasma membranes of some tumor cells and (c) these tumor cells disrupted by shear forces were probably part of cytolysis-conducive rather than of non-cytolysis-conductive conjugates. We combined this approach with the use of cytolysis-inhibiting monoclonal antibodies (mAb), and found that antibody-induced decrease of cytolysis was correlated with a decrease in the percentage of strong or total conjugates, depending on the mAb used. When lectins were added to overcome the inhibitory effect of the mAb, reappearance of cytolytic activity correlated with reappearance of conjugates. This was especially striking using wheat germ agglutinin (WGA): the addition of WGA to irrelevant effector-target cell combinations did not lead to cytolysis; however, the addition of WGA to relevant effector-target cell combinations inhibited by mAb led to reappearance of cytolysis and of strong conjugates. Taken together, these and other results suggested that under our experimental conditions a threshold level of binding strength between effector and target cells might be important, although not sufficient, for T cell-mediated cytotoxicity. These results were not inconsistent with the involvement of mechanical factors in this process. Also, they were in line with the concept of nonantigen-specific lymphoid cell surface interacting molecules, detected by the mAb used and important for the establishment of strong, functional lymphocyte target cell interactions. Finally, they underlined the necessity of a quantitative estimate of cell-cell binding strength when investigating the effect of a given agent (e.g. a mAb) on lymphocyte target cell recognition.     

Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ, and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ. NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122(+)NK1.1(+)DX5(-) stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo. Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.     

T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR‐binding parameters

T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self‐peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide–MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN‐γ production by T cells, and 1G4‐pMHC dissociation rates measured in soluble phase or on surface‐bound molecules, displayed six‐ to sevenfold variation among pMHCs. When T cells were dropped onto pMHC‐coated surfaces, rapid spreading occurred after a 2‐min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all‐or‐none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.     

Rat monoclonal antibodies to mouse IgG1, IgG2a, IgG2b, and IgG3 subclasses, and kappa chain isotypic determinants

Rats of the LOU/Ws1 strain were immunized with mixtures of mouse monoclonal antibodies (MAbs) of various isotypes, and their spleen cells were fused with the rat myeloma Y3.Ag1.2.3 or the mouse myeloma X63.Ag8.653. From four fusion experiments, we have selected 14 rat MAbs that exhibited selective binding to either IgG2a, IgG2b, IgG1, and IgG3 subclasses or to kappa chain isotypic determinants. Cross-blocking studies revealed that three rat MAbs identified distinct determinants on the Fc fragment of the MAb H10-81.10 (A.TH, Igh-1e). By contrast, the IgG1, IgG2b, IgG3, and kappa isotypes defined by the mAbs analyzed in this study were found to be in close spatial relationship. These rat MAbs bound IgG2a, IgG2b, or IgG1 mouse MAbs, expressing the Igh-1e,j,a, or c, Igh-3a or b, or Igh-4a or b allelic specificities, respectively.     

A unique CD44 monoclonal antibody identifies a new T cell activation pathway.

We have identified a new T cell activation pathway mediated by the lymphocyte homing receptor/CD44 molecule, 8B2.5, a local monoclonal antibody (mAb), which recognizes two glycoproteins of 85 and 220 kDa with wide tissue distribution, is shown by sequential immunoprecipitations and competitive antibody-binding inhibition experiments with several CD44 reference mAb to recognize the CD44 molecule. The 8B2.5 mAb, but not reference CD44 mAb, is able to induce resting peripheral blood lymphocytes to proliferate in the presence of phorbol esters. This proliferation is monocyte dependent but Fc independent and results from 8B2.5 mAb binding to CD44 molecules both expressed by both T cells and monocytes. In the absence of monocytes, proliferation can be restored by solid-phase 8B2.5 mAb, or, to a lesser extent, by adding interleukin 2. Although CD3 and CD44 surface molecules are found physically independent, T cell activation via the CD44 pathway is inhibited by CD3 modulation. In addition to the direct role of CD44 molecules in T cell proliferation, CD44 mAb can up- or- down-regulate the CD3 and CD28 pathways, depending on the presence of monocytes. These results suggest that T cell and monocyte binding to high endothelial venule or extracellular matrix proteins could further promote clonal expansion of resting T cells migrating in certain specific anatomic sites.     

A murine anti-I-A monoclonal antibody recognizes human DR (I-E-like) antigens

A monoclonal antibody, H39-49.5, originally raised against the murine I-A^k antigen, recognizes a nonpolymorphic determinant on HLA-DR-like molecules from human cells. These HLA-DR-like molecules have been characterized by amino acid sequencing and have been found to be homologous to the murine I-E antigens rather than the murine I-A antigens. These results suggest a common origin for the murine I-E, I-A, and HLA-DR antigens and caution that serological cross-reactivity alone cannot be used to reliably establish the presence of structural homologues of I-E or I-A-like molecules in other species.