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The natural killer cell (NK)-specific p58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between p58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183-EB6+ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4+ C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183+/EB6+ clones after cell lysis in the presence of digitonin. The p58 receptor also revealed an association with molecules belonging to the zeta family (i.e. CD3 zeta and Fc epsilon RI gamma chains). Two-dimensional diagonal gel analysis of the p58 complex immunoprecipitated from polyclonally activated p58+ NK cells indicated a preferential association with CD3 zeta chains either in the form of covalently linked zeta-zeta homodimers or in the form of zeta-gamma heterodimers, while gamma-gamma homodimers were detectable in low amounts. However, p58+ clones displaying a unique association with gamma-gamma homodimers could also be isolated. Probing the immunoprecipitated p58 complex with anti-p56lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via p58 molecules induced increments of p58/p56lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 zeta chains, no tyrosine phosphorylation was detected in the p58-associated CD3 zeta chains. In these in vivo experiments neither anti-CD16 nor anti-p58 mAb could induce tyrosine phosphorylation of the gamma chains. Finally, the anti-p58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompanied by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 zeta chains.

The surface expression of given HLA class I alleles protects target cells from lysis mediated by natural killer (NK) clones specific for these (or related) alleles. We could define two groups of NK clones specifically recognizing either Cw4 and related C alleles ("group 1") or Cw3 and related C alleles ("group 2"), respectively. Monoclonal antibodies (mAb) to class I molecules should interfere with the interaction between NK receptors and class I molecules, thus resulting in lysis of protected target cells. However, none of the numerous available mAb to class I molecules had this effect. Therefore, we attempted to select new mAb on the basis of their ability to induce lysis of Cw4- or Cw3-protected lymphoblastoid cell lines by "group 1" or "group 2" NK clones, respectively. From mice immunized with phytohemagglutinin (PHA)-activated lymphocytes expressing either Cw3 or Cw4 alleles, two mAb were selected, the 6A4 (IgG1) and the A6-136 (IgM), on the basis of their ability to induce lysis of protected target cell. Both mAb immunoprecipitated molecules which, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gave two bands of 45 and 12 kDa, typical of the class I heavy chain and beta 2 microglobulin, respectively. It has been proposed (but not proven), that self major histocompatibility complex class I molecules protect normal cells from autologous NK cell lysis. Thus, we used the 6A4 and A6-136 mAb to assess this possibility directly. Cw4-specific ("group 1") and Cw3-specific ("group 2") NK clones were isolated from donors expressing the corresponding (or related) protective C alleles. None of these clones lysed autologous PHA-induced blasts, used as target cells. However, addition of the F(ab')2 of 6A4 mAb or the A6-136 mAb resulted in lysis of autologous target cells by "group 1" or "group 2" NK clones, respectively. These data provide direct evidence that the expression of class I molecules protects normal cells from lysis by autologous NK cells.

Two monoclonal antibodies (mAb), termed ED6 and LD6, were obtained by immunizing mice with cytotoxic T cell lines expressing the T cell receptor (TcR) gamma/delta. These mAb were selected according to their ability to trigger the cytolytic program of the immunizing cell lines in a redirected killing assay. Both mAb recognized molecule(s) expressed on the surface of most long-term cultured TcR gamma/delta +, TcR alpha/beta + and CD3-CD16+ lymphocytes, while it was absent on resting peripheral blood lymphocytes. In addition both mAb reacted with neoplastic B cell lines, Epstein-Barr virus-transformed B cell lines, small cell lung cancer and glioma cell lines, while no surface reactivity was detected on ovarian, breast, colon and non-small cell lung cancer lines. The functional activity of these mAb was studied by two cytolytic assays. Both mAb were able to trigger the cytolytic program of CD3+TcR gamma/delta + polyclonal cell lines and of a CD3-CD16+ NK cell clone against the murine mastocytoma target cell line P815 (Fc receptor+) in a 4-h 51Cr-release assay. In addition, ED6 and LD6 hybridomas were lysed by TcR gamma/delta + effector cells while other hybridomas (obtained from the same fusion) were not lysed. ED6 and LD6 mAb (in the presence of submitogenic doses of the phorbol 12-myristate 13-acetate) also induced the secretion of interleukin 2 by ED6/LD6+ T cell clones expressing TcR gamma/delta or alpha/beta. mAb-induced surface antigen modulation experiments showed that the antigenic determinant recognized by ED6 and LD6 co-modulated, thus indicating that the two mAb probably recognize the same or closely associated molecules. The molecular characteristics of the antigen recognized by the mAb were investigated by Western blot analysis. The LD6 mAb recognized a major band of approximately 65 kDa, both under nonreducing and reducing conditions. These data indicate that ED6 and LD6 mAb recognize a novel non-lineage-specific activation antigen which is involved in the induction of the functional program of long-term cultured T or natural killer cells.

The T cell receptor (TcR) beta chain is encoded by a 1.3-kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions. A shorter 1.0-kb transcript with C but no V sequences is found in thymocytes, alpha/beta and gamma/delta T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0-kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and gamma/delta lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) "J-C clones" consisting of one of five J beta regions and the corresponding C beta 1 or C beta 2 regions; (b) "D-J-C clones" composed of D beta 1/or D beta 2/J beta rearranged sequences and C beta 1 or C beta 2 sequences; (c) "5'C clones" made up of C beta 1 or C beta 2 regions preceded by the corresponding genomic 5' flanking region. The presence of recombination signal sequences in J-C and D-J-C clones suggests that the first derive from mRNA transcribed from promoters located in the 5' J region and the second from mRNA transcribed from promoters situated in the 5' D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic T beta protein with a variable D-J beta N terminus. These findings have implications for the mechanisms of T beta germ-line transcription and the function of the T beta transcripts.

Investigations on T cell receptor delta chain (TcR delta) gene expression in freshly isolated normal large granular lymphocytes, natural killer (NK) cell clones and NK bulk cultures revealed six T delta transcripts (3.5, 3.1, 2.2, 2.0, 1.5 and 1.3 kb) which varied in number and relative abundance in the different samples. None of the six known T delta variable regions was expressed and the T delta locus was retained in its germ-line configuration. The origin and significance of these NK-associated T delta transcripts are discussed.

A human T cell receptor (TcR) delta chain variable region segment, V delta 3, has been mapped 2 kilobases 3' to the constant (C delta) gene. The V delta 3 gene segment is in an inverted transcriptional orientation to the diversity regions (D delta), joining regions (J delta) and C delta gene segments. Analyses of TcR delta cDNA clone, KT041, showed that V delta 3 is linked to D delta 1, D delta 2, J delta 3 and C delta and that this message encodes a potentially functional TcR delta chain. These findings indicate that a functional TcR delta gene can be constructed through a chromosomal inversion, suggesting that in human, chromosomal inversion is one of the important rearrangement mechanisms.

We have applied two-color fluorescence cytofluorometric techniques to the analysis of the distribution of T44 and CD3 antigens in peripheral blood human lymphocytes. While most CD3+ cells co-expressed T44 antigen, a small distinct subset was CD3+ T44- (2-10% of CD3+ cells). This cell subset also did not react with the WT31 monoclonal antibody (mAb), specific for an alpha/beta framework determinant of the T cell receptor (TCR). Lack of T44 antigen expression was also observed in purified CD3+ WT31- polyclonal populations that had been cultured in medium containing interleukin 2 (IL2) and as well as greater than 30 clones expressing the CD3+4-8-WT31- surface phenotype. Immunoprecipitation experiments confirmed that expression of T44 molecules was confined to CD3+ WT31+ peripheral blood T cells. While conventional CD3+ WT31+ cells produced IL2 in response to mAb directed to CD2, CD3 or T44 surface molecules, CD3+ WT31- cells did not respond to anti-T44 mAb but released IL2 following stimulation with anti-CD2 or anti-CD3 mAb. Therefore, assuming that anti-T44 mimicks the effect of a still undefined natural ligand our data suggest that T cells expressing the gamma-gene surface product may be signalled by stimuli which differ, at least in part, from those acting on CD3+ WT31+ T lymphocytes.

Alloreactive clones expressing T cell receptor (TcR) gamma/delta were derived by limiting dilution from CD3+ CD4- CD8- WT31- populations stimulated in allogeneic mixed lymphocyte culture. These clones specifically lysed phytohemagglutinin-induced blast cells bearing the stimulating alloantigens, whereas they had no effect on autologous or allogeneic unrelated target cells. Analysis of the reactivity with monoclonal antibodies (mAb) specific for two different subsets of TcR gamma/delta (BB3 and delta-TCS-1) showed that five out of nine clones were BB3+, whereas the remaining reacted with delta-TCS-1. Therefore, we can conclude that both subsets of TcR gamma/delta+ cells are able to specifically recognize and lyse allogeneic cells. mAb directed against the CD3-TcR gamma/delta molecular complex strongly inhibited the specific cytolytic activity of TcR gamma/delta+ clones, whereas they had no effect on the lysis of the natural killer-sensitive K-562 target cells mediated by the same clones. An alloreactive delta-TCS-1+ clone (LM12) was further characterized for its specificity. LM12 clone had been derived after stimulation in mixed lymphocyte culture against donor M.M. (HLA typing: Aw68, 24; B35, w55; DR1, 7). The analysis of a large panel of phytohemagglutinin-induced target cells revealed that only the HLA-A24+ target cells were lysed. The direct evidence that the A24 molecule represented the restriction element was provided by experiments using A24-transfected murine P815 target cells. Thus, clone LM12 efficiently lysed A24-transfected P815 cells, but not the same cells untransfected or transfected with the Cw3 gene. Therefore, it appears that polymorphic determinants of class I major histocompatibility complex molecules can be the target of TcR gamma/delta+ alloreactive cell recognition.

The small peripheral blood CD3+ T cell population lacking both CD4 and CD8 surface antigens has been analyzed in the present study. Enriched CD3+4-8- populations were obtained by depletion with anti-CD4 or anti-CD8 monoclonal antibodies (mAb) and complement. The resulting populations contained greater than 99% CD2+ cells, whereas CD3+ represented approximately 50%. Virtually all of the cells were CD4-8- and did not react with the WT31 mAb, specific for a framework determinant of the alpha/beta T cell receptor (TCR). In order to analyze the molecular nature of CD3-associated molecules in CD3+WT31- populations, cells were stimulated with 0.5% phytohemagglutinin (PHA) for 24 h and expanded for an additional 7-14 days in interleukin 2 (IL 2). The resulting cells were greater than 95% CD3+ and expressed neither CD4/CD8 nor WT31 antigen. Cell surface iodination followed by cross-linking and immunoprecipitation with anti-CD3 mAb showed that CD3-associated molecules consisted of a major 45-kDa band and a minor band of 43 kDa. Thus, whereas CD3-associated molecules isolated from polyclonal CD3+WT31+ populations (expanded in IL 2 under the same culture conditions) appeared as diffuse bands, CD3-associated molecules isolated from CD3+WT31- populations displayed a homogeneous molecular mass. Northern blot analysis revealed the presence of mRNA for the TCR gamma chain whereas the mRNA for the alpha chain was mostly represented by a truncated (1.2 kb) form. Also small amounts of a nonproductive mRNA for the beta chain were detected. Freshly isolated CD3+WT31--enriched populations proliferated in response to PHA and concanavalin A, moreover, IL 2 was detected in the culture supernatants after cell stimulation. By applying culture conditions which allow virtually all T cells to undergo clonal expansion, approximately 1/3 CD3+WT31- were clonogenic. In addition, the large majority of proliferating microcultures lysed the K562 cell line and about half the natural killer (NK)-resistant fresh melanoma target cells. A large number of clones derived from CD3+WT31- enriched populations by limiting dilution has been further analyzed. More than 95% of the clones were CD3+4-8-WT31-; 12/15 clones analyzed in more detail displayed NK activity and 6/15 lysed melanoma cells; in addition, all lysed P815 target cells in the presence of PHA, thus indicating that all the clonogenic CD3+WT31- cells have a cytolytic potential.

A polyclonal CD3+4-8-WT31- cell line (termed SFG) was utilized for mice immunization in order to produce monoclonal antibodies (mAb) specific for the T cell receptor (TcR) gamma/delta. Hybrid supernatants were screened for their ability to induce SFG cells (but not conventional TcR alpha/beta + CTL lines) to kill the murine Fc receptor-positive P815 target cell line. Three hybrids, termed G1, A13 and F11, were isolated according to this screening. By indirect immunofluorescence G1 mAb reacted with 65% of SFG cells, while A13 stained 26% and F11 75% of cells. Double-fluorescence analysis revealed that G1 and A13 mAb identify two distinct, non-overlapping subsets of cells present in the SFG cell line. The reactivity of the mAb was also analyzed on a panel of representative TcR gamma/delta clones. G1 mAb reacted with 5 clones, that were also stained by the previously described BB3 mAb (recognizing the disulfide-linked form of TcR gamma/delta). These clones failed to react with A13 and delta-TCS-1 mAb (the latter of which is known to react with a non-disulfide-linked form of TcR gamma/delta). Out of six clones that reacted with A13 mAb, four were also delta-TCS-1+, whereas two were delta-TCS-1- and none of them reacted with G1, (or BB3) mAb. In contrast to the mAb above, F11 brightly stained the G1+A13- clones and more weakly the G1-A13+ clones. Moreover, F11 efficiently triggered both types of clones to kill the P815 target cells while G1 and A13 were able to trigger only G1+ or A13+ clones, respectively. None of the mAb above reacted with a large number of CD3+WT31+ clones. Antibody-induced surface antigen modulation experiments indicated that molecules recognized by G1, A13 and F11 were physically associated on cell surface with CD3 determinants. In addition, immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (performed on 125I-surface-labeled TcR gamma/delta+ clones) revealed that molecules recognized by G1, A13 and F11 displayed an apparent mol. wt. corresponding to that of CD3-associated TcR molecules, immunoprecipitated by anti-CD3 mAb from the same clones.