The 1A4 molecule (CD27) is involved in T cell activation.

We developed a new mAb, anti-1A4, which recognizes an epitope on the CD27 molecule distinct from those recognized by several known anti-CD27 mAb. Although it has been suggested that the CD27 molecule is a T cell activation Ag, there was little direct evidence that the structure was involved in the T cell activation process. In this study, we showed that anti-1A4 inhibited anti-CD2, anti-CD3, mitogens, or soluble Ag-induced T cell proliferation as well as PWM-driven B cell IgG synthesis. Interestingly, anti-1A4 inhibited IL-2 secretion without affecting IL-2R expression. In addition, pretreatment of T cells with anti-1A4 inhibited the normally sustained intracellular calcium mobilization seen after triggering of T cells via the CD2 or CD3 pathways. Thus, binding of anti-1A4 to the CD27 molecule appears to induce a negative effect on T cell activation. This may be due to either a direct signal to T cells or the blocking of an interaction between T cells and accessory cells or both. These findings support the notion that the CD27 molecule plays an integral role in the process of T cell activation.     

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.     

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.     

Differential regulation of Ca2+ mobilization in human thymocytes by coaggregation of surface molecules.

Variations in intracellular Ca2+ levels in developing thymocytes are likely to play a major role in both the activation-associated differentiation of thymocytes and in the selection or clonal deletion of cells. Here we examine the role of CD4, CD8, CD2, and CD45 in the regulation of intracellular Ca2+ levels in mature and immature thymocytes. Mature and immature thymocytes, distinguished on the basis of their CD5 expression, were analyzed simultaneously for their ability to mobilize Ca2+ after coaggregation of their CD3/TCR with other thymic surface Ag. Flow cytometric analysis by using Indo-1 showed that coaggregation of CD4, CD8, and CD2 with CD3/TCR clearly enhances a minimal signal delivered via CD3/TCR on immature thymocytes. Coaggregation with class I MHC had no discernible effect. The responsiveness of immature thymocytes correlated strictly with CD3 surface expression, such that loss of responsiveness occurred with reduced CD3 cell-surface density. However, even thymocytes with very low CD3 expression were able to respond to triggering via CD3 under optimal conditions, indicating that the CD3 signal-transducing mechanism is functional on early thymic cells. Intracellular increases in Ca2+ concentrations induced via CD3, could effectively be inhibited by cross-linking of CD45 and CD3 on immature thymocytes. Although triggering via CD2 alone induced a strong Ca2+ flux, prolonged incubation with activating anti-CD2 antibodies made thymocytes refractory to subsequent triggering. Refractoriness was associated with partial loss of surface CD3 and CD3 zeta. Our results indicate that thymic surface Ag are differentially involved in the regulation of intracellular Ca2+ levels in immature as well as mature thymocytes.     

A monoclonal antibody reactive with a 15-kDa cytoplasmic granule-associated protein defines a subpopulation of CD8+ T lymphocytes

We have recently described a novel method for the production and characterization of mAb reactive with T cell-restricted intracellular antigens. From a panel of antibodies that react specifically with permeabilized T lymphocytes but not with permeabilized B lymphocytes or native T cells, we have selected one, designated TIA-1, that reacts with 20 to 36% of digitonin permeabilized peripheral blood T lymphocytes. Flow cytometric analysis of purified CD4+ and CD8+ subsets showed TIA-1 to recognize a subpopulation of 49 to 64% of CD8+ lymphocytes. Little or no reactivity with CD4+ resting T lymphocytes was observed. TIA-1 did not react with any of a panel of T cell lines, B cell lines, or monocytoid cell lines. TIA-1 reacted strongly with NK cell clones and CD8+ cytolytic T cell clones, and less strongly with CD4+-activated T cell clones, suggesting a preferential expression in cells possessing cytolytic potential. Cell fractionation experiments showed TIA-1 to be membrane associated. Furthermore, Percoll gradient fractionation of a cytolytic T cell clone (T4T8C1) showed the majority of TIA-1 to be contained in a low density membrane fraction that also contained serine protease activity. Immunoelectron microscopy showed TIA-1 to decorate the membranes of electron lucent and electron dense cytoplasmic granules in this same cytolytic T cell clone. Biochemical analysis showed TIA-1 to be a 15-kDa protein in unstimulated T cells. Upon activation with Con A or anti-CD3 antibodies. TIA-1 was induced to form disulfide linked dimers, trimers, and tetramers of the basic 15-kDa unit. Taken together, our data suggest that TIA-1 is a cytolytic granule associated protein that may define a subpopulation of resting CD8+ T lymphocytes possessing cytolytic potential.     

The 2H4 molecule but not the T3-receptor complex is involved in suppressor inducer signals in the AMLR system

It is suggested that autologous mixed lymphocyte reaction (AMLR) may play an important role in generating suppressor inducer signals and in down-regulating the immune response following self-major histocompatibility recognition. In the present study, monoclonal antibodies directed at cell surface structures on T4+ cells activated in AMLR were used to define the molecules important in the generation of the suppressor inducer signal. The density of a 200/220-kDa structure, termed 2H4, increased on T4 cells during activation in AMLR and furthermore a strong correlation was observed between the generated suppressor inducer activity of such cells and the density of the 2H4 antigen. More importantly, we showed that treatment of AMLR activated T4 cells with anti-2H4 but not anti-T3 or T4 antibody abolished the suppressor inducer function of these cells. These results suggest that the 2H4 molecule but not the T3-receptor complex plays an important role in generating suppressor inducer signals in the AMLR system.     

The role of interleukin 2 and T11 E rosette antigen in activation and proliferation of human NK clones

Although considerable data have recently been accumulated regarding the functional role of natural killer (NK) cells, relatively little is known about the factors that regulate NK cell activity. In these studies, we evaluated the role of interleukin 2 (IL 2) and the expression of the IL 2 receptor in the activation and proliferation of human NK cloned cell lines. By using a series of cloned cell lines, we were able to analyze homogeneous populations of NK cells that ordinarily comprise only a small fraction of peripheral blood lymphocytes and are extremely heterogeneous with respect to phenotypes and cytotoxic specificities. In comparison with several T cell clones, we found a much lower density of IL 2 receptors on NK clones, regardless of whether or not these cloned cells had a mature T cell phenotype. Correspondingly, NK clones needed a 10-fold higher concentration of recombinant IL 2 for maximal proliferation. Moreover, blocking studies with specific monoclonal IL 2 receptor antibodies indicated that IL 2 is both necessary and sufficient to induce the proliferation of NK clones. Because the majority of peripheral blood NK cells and NK clones express the T11 E rosette receptor antigen, which has been shown to be an antigen-independent activation pathway for T cells, we were able to study the role of monoclonal anti-T11 antibodies in the activation of various NK clones for which a specific target antigen is not known. In contrast to T cell clones, the induction of IL 2 receptor expression after T11 activation was possible only for some NK clones such as JT10 and JT3, but not for CNK5. Before activation, the IL 2 receptor expression of NK clones was confined to cells in the G2 - M phase, but after T11 activation the more pronounced IL 2 receptor expression became independent of the cell cycle. With respect to the direct proliferative effect of anti-T11 activation that has been noted with T cell clones, only the T3+ (JT10) and not the T3- NK clones could be directly stimulated. Nevertheless, IL 2 receptor expression could be triggered on some T3- clones such as JT3. Because T11-induced proliferation of T cells has been shown to be dependent on both the expression of the IL 2 receptor and on the interaction of this receptor with IL 2, it is proposed that the different responses of NK cells to T11 activation may reflect the ability of the individual clone to produce endogenous IL 2, as well as its ability to express the IL 2 receptor.     

Expression of a 26,000-dalton glycoprotein on activated human T cells

In the present study, we describe the expression of a 26,000-dalton glycoprotein on human T cells following stimulation by either mitogen or alloantigen. This glycoprotein, which is the target antigen of a monoclonal antibody designated J2, is distinct from Ia-like molecules and is not present on resting T cells. We demonstrate GP 26 expression in both major immunoregulatory subsets i.e., T4⁺ (inducer) and T8⁺ (cytotoxic/suppressor) following activation and show that the GP 26-bearing cells are not directly responsible for the cytotoxicity generated in MLR. The relationship of the J2 target antigen to other glycoproteins with similar molecular weight which have been described on activated T cells is discussed.     

Essential role of caveolae in interleukin-6- and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple myeloma cells

Caveolae, specialized flask-shaped lipid rafts on the cell surface, are composed of cholesterol, sphingolipids, and structural proteins termed caveolins; functionally, these plasma membrane microdomains have been implicated in signal transduction and transmembrane transport. In the present study, we examined the role of caveolin-1 in multiple myeloma cells. We show for the first time that caveolin-1, which is usually absent in blood cells, is expressed in multiple myeloma cells. Analysis of myeloma cell-derived plasma membrane fractions shows that caveolin-1 is co-localized with interleukin-6 receptor signal transducing chain gp130 and with insulin-like growth factor-I receptor. Cholesterol depletion by beta-cyclodextrin results in the loss of caveola structure in myeloma cells, as shown by transmission electron microscopy, and loss of caveolin-1 function. Interleukin-6 and insulin-like growth factor-I, growth and survival factors in multiple myeloma, induce caveolin-1 phosphorylation, which is abrogated by pre-treatment with beta-cyclodextrin. Importantly, inhibition of caveolin-1 phosphorylation blocks both interleukin-6-induced protein complex formation with caveolin-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. beta-Cyclodextrin also blocks insulin-like growth factor-I-induced tyrosine phosphorylation of insulin-responsive substrate-1 and downstream activation of the phosphatidylinositol 3-kinase/Akt-1 pathway. Therefore, cholesterol depletion by beta-cyclodextrin abrogates both interleukin-6- and insulin-like growth factor-I-triggered multiple myeloma cell survival via negative regulation of caveolin-1. Taken together, this study identifies caveolin-1 and other structural membrane components as potential new therapeutic targets in multiple myeloma.     

Generation of CD8 (T8) cytotoxic cells has a preferential requirement for CD4+2H4- inducer cells

CD8 (T8) cells are capable of both suppression and cytotoxicity. However, we have found that the activation of CD8 cytotoxic cells has a preferential requirement for a different CD4 (T4) subset from that previously reported for the activation of CD8 suppressor cells. We have recently characterized two monoclonal antibodies which subdivide CD4 cells into inducers of help for antibody production (CD4+ 4B4+) and inducers of CD8 mediated suppression (CD4+2H4+). We now report that CD4+4B4+2H4- cells also preferentially induce CD8-mediated cytotoxicity. Human peripheral blood T cells were fractionated into CD8, CD4, CD4+2H4+, and CD4+2H4- populations by both the adherence to antibody-coated plates and the fluorescence-activated cell sorter. The cells were cultured 6 days with irradiated allogeneic non-T cells and a cytotoxicity assay was then performed using cryopreserved non-T cells as targets. It was found that the combination of CD4+2H4- cells and CD8 cells resulted in greater cytotoxicity than either CD4 + CD8, or CD4+2H4+ + CD8. The combination of CD4+2H4+ cells with CD8 cells resulted in minimal cytotoxicity, which was similar to that generated by CD8 cells alone. These results were confirmed using anti-4B4 to positively select the reciprocal CD4 subset. Furthermore, the cytotoxicity induced by CD4+2H4- cells was alloantigen specific and Class I major histocompatibility complex restricted. As both CD4+2H4+ and CD4+2H4- cells proliferate equally well to alloantigen and produce similar levels of interleukin 2 (IL-2), it is likely that the generation of CD8 cytotoxic cells requires a signal in addition to IL-2.