T cell regulation of myelopoiesis: analysis at a clonal level

Colony-stimulating factor (CSF) production by a series of cloned human T lymphocyte cell lines was examined by substituting cloned T cells for peripheral blood mononuclear cells in the feeder layer of a double-layer agar CFU-C assay system. Of 12 T cell lines tested, all produced CSF when stimulated by specific antigen, whereas CSF production in the absence of stimulation was generally negligible. In the case of soluble antigen-specific (ragweed or tetanus toxoid) clones, this required both nominal antigen and the appropriate MHC gene product on autologous antigen-presenting cells, whereas in the case of clones specific for EBV-transformed B cell lines (allogeneic or autologous), surface-bound EBV-related antigen and MHC was necessary. When tested in this manner, CSF production by different cloned T cells was heterogeneous in both amount and subclass. Thus, although most clones stimulated growth of granulocyte, monocyte, and eosinophil colonies, certain clones were identified which preferentially stimulated some colony types but not others. This heterogeneity was particularly evident with respect to eosinophil colony production. In addition, a soluble inhibitor of granulocyte colony growth was produced by one clone. These findings provide further support for the notion that antigen-specific T cells may, on activation, regulate myelopoiesis in a precise way, and provide a possible cellular basis for selective eosinophilia, monocytosis, or neutrophilia seen in certain disease states.     

Mechanism of annexin I-mediated membrane aggregation

It has been proposed that annexin I has two separate interaction sites that are involved in membrane binding and aggregation, respectively. To better understand the mechanism of annexin I-mediated membrane aggregation, we investigated the properties of the inducible secondary interaction site implicated in membrane aggregation. X-ray specular reflectivity measurements showed that the thickness of annexin I layer bound to the phospholipid monolayer was 31 +/- 2 A, indicating that annexin I binds membranes as a protein monomer or monolayer. Surface plasmon resonance measurements of annexin I, V, and mutants, which allowed evaluation of membrane aggregation activity of annexin I separately from its membrane binding, revealed direct correlation between the relative membrane aggregation activity and the relative affinity of the secondary interaction site for the secondary membrane. The secondary binding was driven primarily by hydrophobic interactions, unlike calcium-mediated electrostatic primary membrane binding. Chemical cross-linking of membrane-bound annexin I showed that a significant degree of lateral association of annexin I molecules precedes its membrane aggregation. Taken together, these results support a hypothetical model of annexin I-mediated membrane aggregation, in which a laterally aggregated monolayer of membrane-bound annexin I directly interacts with a secondary membrane via its induced hydrophobic interaction site.     

The physiological basis of seed dormancy in Avena fatua. II. On the involvement of alternative respiration in the stimulation of germination by sodium azide

Chromatography on DEAE-cellulose of an extract from etiolated leaves of sorghum ( Sorghum vulgare Pers. cv. INRA 450), a C₄ plant, gave only one form of phosphoenol pyruvate carboxylase with functional and regulatory properties of a C₃ type plant enzyme. Greening of the leaves resulted in a significant increase in activity. This increase was due to the appearance of a new form of the enzyme, which eluted at lower ionic strength and exhibited new properties. This form was glucose-6-P activated and showed a sigmoidal curve response to the concentration of the substrate phosphoerralpyruvate. These kinetic properties are typical of a C₄ plant enzyme.     

Human macrophage-lymphocyte interaction in proliferation to soluble antigen: II. Characterization of lymphocyte binding to antigen-pulsed macrophage monolayers

Human peripheral blood T lymphocytes from individuals immune to tetanus toxoid (TT) or mumps soluble antigen can be depleted of cells capable of proliferating in response to these antigens by specific adsorption to antigen-pulsed macrophage (Mφ) monolayers. Specific adsorption is manifest by depletion of proliferative activity (measured by tritiated thymidine incorporation) in the fraction of T lymphocytes nonadherent to Mφ monolayers pulsed with the appropriate antigen. The occurrence of T lymphocyte-Mφ binding is supported by experiments which failed to detect cell-free factors or cells capable of inhibiting lymphocyte proliferation in the nonadherent lymphoid population. A quantitative analysis of the immunoadsorption assay suggests that proliferative activity representing at least 90% of that displayed by reciprocal control lymphocytes can be depleted on Mφ monolayers. Experiments employing varying numbers of adsorbing Mφ relative to T lymphocytes have established practical limits which predict the efficiency of adsorption. Kinetic studies indicate that specific adsorption occurs within 1 hr, with maximal levels or binding evident by 4–6 hr; binding remains stable for at least 16 hr. Specific adsorption is temperature dependent, occurring maximally at 37 °C, but not at all at 4 °C. T lymphocyte-Mφ interaction is complex and includes more than a recognition of pulsed antigen alone. This conclusion is supported by the finding that antigen-pulsed Mφ fail to absorb MLC-nonidentical allogeneic lymphocytes and that a heterologous antiserum directed against human Ia-like molecules (p23,30) inhibits specific T cell-Mφ binding.     

DRw antisera react with activated T cells.

Nylon wool-purified T cells appear to be nonreactive in a lymphocytotoxicity assay with HLA-DRw antisera and complement before cell activation. However, after activation in mixed lymphocyte culture, responder cells express determinants that are strongly reactive with DRw alloantisera after 6 days and gradually disappear by 16 to 18 days. Restimulation of the primed cells resulted in re-expression of the blast determinants. Mitogenic stimulation with Con A or purified PHA (HA-17) also resulted in temporary expression of these determinants; reactivity usually conformed to DRw genetic restriction; however, occasional extra reactions occurred that were variable depending on the method of activation (i.e., MLC, Con A, or HA-17). The results suggest the presence of additional allospecificities within some of the DRw antisera that react with "Ia-like" antigens on activated cells from unique subsets of T cells. Whether these DRw antisera contain antibodies against T cells or agains activation or differentiation T cell antigens is not as yet clear.     

Activation of human cytolytic cells through CD2/T11. Comparison of the requirements for the induction and direction of lysis of tumor targets by T cells and NK cells

We studied the mechanisms whereby human T cells and NK cells are activated and directed to lyse tumor targets through the CD2 (T11/E-rosette) Ag. Using two cloned NK lines, we showed that these cells, as had previously been shown for T cells, could be directed to lyse an "NK-resistant" tumor target in the presence of antibody heterodimers. These heterodimers consisted of a (mAb) to CD2 (anti-T11(2) or anti-T11(3] linked to a mAb recognizing the tumor cell (J5, anti-CALLA). However, distinct differences between NK cells and T cells were observed with regard to the requirements for such directed lysis: first, only one epitope of CD2 on NK cells (either T11(2) or T11(3] needed to be recognized by the antibody heterodimer in order for directed lysis to occur, whereas for T cells both T11(2) and T11(3) epitopes had to be recognized. Second, in confirmation of previous data with monomeric anti-T11(2) or anti-T11(3) antibody, heterodimers constructed with these reagents enhanced conjugate formation between NK cells and tumor targets, whereas no such enhancement was seen with T cells. All types of heterodimer directed lysis were dependent on the adhesion molecule LFA-1, as an anti-LFA-1 antibody-blocked lysis. Third, whereas in T cells lysis mediated through CD2 appeared to be regulated by CD3 but not vice versa, all types of lysis by NK cells appeared to be regulated through CD2. Finally we showed that F(ab')2 fragments of the anti-T11(2) and anti-T11(3) antibodies could activate NK cells, but were unable to activate T cells either as cloned cytolytic lines, or in populations of PBL. The implications of our findings with regard to the role of CD2 in the activation of cytolytic cells is discussed.     

Evidence for specific association between class I major histocompatibility antigens and the CD8 molecules of human suppressor/cytotoxic cells

Human T lymphocytes, metabolically labeled with 35S-cysteine and 35S-methionine, were reacted with the homobifunctional cross-linking reagent, dithiobis (succinimidyl propionate) (DSP). When detergent lysates from these cells were immunoprecipitated with a monoclonal antibody reactive with the CD8 antigen, a radiolabeled protein of approximately 44 kd was coprecipitated with the CD8 molecule. Immunoprecipitates from detergent lysates prepared without prior chemical cross-linking contained only the 33 kd CD8 molecule. Similar results were obtained when T lymphocytes or a cytotoxic T cell clone (T4T8Cl) were radiolabeled with 32P-orthophosphoric acid. The 44 kd CD8-associated protein was identified as the heavy chain of the class I major histocompatibility antigen by depletion in preclearing experiments with anti-class I MHC antibody and by peptide mapping. Further analyses indicated that the CD8-class I MHC association is due, in part at least, to disulfide bonding, which may be susceptible to cleavage during processing of cell lysates.     

2H1--a novel antigen involved in T lymphocyte triggering

A mAb anti-2H1, was produced against PHA-activated T cells from a lower primate, Aotus trivirgatus. Anti-2H1 reacted with 90% of peripheral T cells but was found to react with only 10% of thymocytes and some, but not all, leukemic T cell lines. 2H1 expression was dramatically increased when thymic cells were activated by Con A plus PMA. In contrast, anti-2H1 did not react with B cells, macrophages, null cells, or hematopoietic stem cell lines. More importantly, anti-2H1 antibody can induce T cell activation and proliferation in synergy with PMA or anti-T11(3). SDS-PAGE analysis of polypeptides immunoprecipitated with anti-2H1 showed two major polypeptides of 140 and 105 kDa. Thus, the 2H1 Ag can be distinguished from T3, T11, and 9.3 Ag. These results indicate that the 2H1 Ag appears to be involved in the activation of T lymphocytes.     

Expression of MY7 antigen on myeloid precursor cells

A murine monoclonal antibody (anti-MY7) has been developed that detects an antigen expressed by 6% of normal human bone marrow cells, including approximately 40% of myeloid colony-forming cells (CFU-C). The number of bone marrow cells and CFU-C expressing MY7 is significantly increased in regenerating bone marrow, but less than 5% of peripheral blood CFU-C express the MY7 antigen. Erythroid precursors are MY7 negative from peripheral blood and bone marrow. Thymidine suicide studies indicate that CFU-C in S-phase tend to be MY7 positive while CFU-C not in S-phase are MY7 negative. MY7 expression thus appears to identify a fraction of CFU-C that is actively proliferating.     

Direct demonstration of the human suppressor inducer subset by anti-T cell antibodies

Prior studies indicated that sera of patients with active juvenile rheumatoid arthritis (JRA) contain anti-T cell antibodies reactive with the T4+ inducer population. More important, depletion of this T cell subset with JRA anti-T cell antibodies (JRA+ T cells) and C abrogated T5/T8+ suppressor T cell function. In the present study, we utilized Ig-coated plate techniques and JRA anti-T cell antibodies to fractionate the T4+ population into T4+JRA+ and T4+JRA- subsets and characterize the individual T4+ inducer subset. It was shown that whereas only the T4+JRA- population responded maximally to the soluble antigens, TT and mumps, both T4+JRA+ and T4+JRA- subsets proliferated equally well to mitogens and alloantigens. Furthermore, B cell immunoglobulin production induced by T4+JRA- T cells was approximately twice that induced by the reciprocal T4+JRA+ subset. In contrast, the T4+JRA+ subset alone activated T8+ T cells to become suppressor effector cells. These results suggest that the T4+JRA+ subset is the inducer of suppressor subpopulation whereas the T4+JRA- subset functions maximally as the inducer of B cells. It is believed that the suppressor inducer population may have a central role in the immunoregulatory network in man.