CD16 on human gamma delta T lymphocytes: expression, function, and specificity for mouse IgG isotypes.

We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3- CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.     

Cytotoxic T lymphocyte triggering via CD16 is regulated by CD3 and CD8 antigens. Studies with T cell receptor (TCR)-alpha beta+/CD3+16+ and TCR-gamma delta+/CD3+16+ granular lymphocytes

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.     

CD4-CD8- human T cells: phenotypic heterogeneity and activation requirements of freshly isolated "double-negative" T cells

In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C'-mediated lysis and panning. While the majority (79.0 +/- 12.0%) of DN cells were TCR gamma delta+ as shown by staining with mAb TCR delta-1, a minor fraction (6.7 +/- 4.7%) expressed TCR alpha beta as revealed by staining with mAb BMA031. Within the gamma delta+ DN fraction, most cells reacted with mAb Ti gamma A which delineates a V gamma 9JPC gamma 1 epitope, whereas a minor fraction stained with mAb delta TCS-1 which identifies a V delta 1J delta 1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIL-2 and/or rIl-4) are provided.     

NK-like cytotoxicity of allospecific gamma delta TCR+ T cell clones]

We recently generated a series of human alloantigen-specific, CD3+, gamma delta- TCR+ clones by stimulating CD3+, CD4-, CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). These clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Most but not all of these clones express the NK cell associated marker, CD57, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not NK-resistant line, Raji. Gamma delta clones which lacked expression of CD57 had no detectable NK activity. The allospecific cytotoxicity of CD57+ and CD57- clones was inhibited by mAb to CD3 or the TCR delta- chain. In contrast, the NK-like activity of the CD57+ clones was enhanced by these antibodies over a wide range of antibody concentration. An HLA class I framework-specific mAb had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the gamma delta- TCR+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+, TCR- gamma delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

CD3Ti+ human thymocyte-derived clones displaying a differential response to activation via CD3Ti and CD2

Previous studies indicated that, unlike peripheral T-cells, freshly isolated thymocytes show little or no proliferation to activation signals via either the antigen/MHC receptor complex (CD3Ti) or the CD2 structure, unless exogenous IL-2 or phorbol esters are added. To investigate these differences in more detail, we have studied the response of clonal populations of mature thymocyte subsets as well as peripheral T-cell clones to activation via either CD3Ti or CD2. Here we report the characterization of three clones belonging to different subsets of mature thymocytes: CD3+ CD4+ (Ti alpha/beta), CD3+ CD8+ (Ti alpha/beta), and CD3+ CD4- CD8- (Ti gamma/delta). All three clones could be induced to proliferate to insolubilized anti-CD3 mAb. In contrast, activating anti-CD2 mAbs, which induced proliferation in all peripheral T-cell clones tested, did not induce an appreciable proliferation of the thymocyte clones. The latter required additional signals provided by the phorbol ester PMA. However, anti-CD2 mAbs were able to induce early activation events such as phosphoinositide turnover and [Ca2+]i increase to an extent similar to the ones elicited by anti-CD3 mAb. These results further support previous findings suggesting that mature thymocytes are not functionally identical to peripheral T-cells.     

Functional reactivity of WT31 monoclonal antibody with T cell receptor-gamma expressing CD3+4-8- T cells

About 3% of normal peripheral blood T lymphocytes have the phenotype CD3+4-8-. The vast majority of these cells lack the conventional TCR-alpha-, beta complex but express the recently identified TCR-gamma, delta/CD3 receptor complex. These TCR gamma+/CD3+ cells were initially discovered by using as a criterion the lack of reactivity with WT31 mAb. This mAb has been reported to recognize a "framework" epitope on the TCR-alpha, beta/CD3 complex. However, using high concentrations of WT31 mAb, low levels of reactivity with the cell membrane of TCR-gamma+ cells can be observed. This reactivity was significantly increased upon removal of sialic acid residues by neuraminidase. In addition, WT31 mAb is capable to induce lysis by TCR-gamma+ clones. Moreover, immunoprecipitation with WT31 by using cell lysates prepared with the mild detergent digitonin resulted in the isolation of the intact TCR-gamma/CD3 complex. Thus, in contrast to what was previously assumed, WT31 mAb also reacts with a functional epitope present on gamma, delta/CD3 T cells, and therefore lack of reactivity with WT31 mAb is not always a proper hallmark for TCR-gamma-expressing cells.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Activation requirements of newborn thymic gamma delta T cells.

We have analyzed the requirements for the induction of proliferative responses by thymic CD4-CD8- gamma delta T cells. Enriched populations of CD4-CD8- thymocytes from newborn mice, purified by negative selection with anti-CD4, anti-CD8, and anti-TCR alpha beta mAbs were found to contain approximately 20% gamma delta T cells that were p55IL-2R-. When these cells were cultured with a panel of lymphokines (IL-1, -2, -4, and -7), a small response was observed to some of the cytokines tested individually; however, combinations of certain lymphokines (IL-1 + 2, IL-1 + 7, and IL-2 + 7) were found to induce significant proliferation and the selective outgrowth (75-90%) of gamma delta T cells. These cells were IL-2R+, remained CD4-, yet expressed variable levels of CD8. A limited analysis with specific anti-V gamma and V delta mAb suggested that there had not been a selective expansion of preexisting V gamma 2, V gamma 3, or V delta 4 populations in response to the stimulatory lymphokine combinations. Thymic CD4-CD8- gamma delta T cells were unresponsive to stimulation with immobilized anti-pan gamma delta mAb alone. However, in the presence of immobilized anti-pan gamma delta mAb and IL-1, IL-2, or IL-7, but not IL-4, a vigorous proliferative response was observed. Phenotypic analysis showed that 80 to 95% of the proliferating cells were polyclonally expanded gamma delta T cells, expressed the p55IL-2R, and the majority remained CD4-CD8-. Blocking studies with anti-IL-2R mAb showed that stimulation with anti-pan gamma delta + IL-1, but not anti-pan gamma delta + IL-7 was dependent on endogenously produced IL-2. Collectively, these studies suggest that the activation requirements of newborn thymic gamma delta T cells differ markedly from alpha beta T cells in that gamma delta T cells 1) respond to combinations of cytokines in the absence of TCR cross-linking, 2) can respond to TCR cross-linking in the presence of exogenous cytokines, 3) but are unable to activate endogenous cytokine production solely in the presence of TCR cross-linking.     

Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cell-mediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes

To evaluate the capability of NK cells and cytotoxic T lymphocytes to interact with normal hematopoietic progenitor cells (HPC), as compared to neoplastic lymphohematopoietic cells, we investigated inhibition of colony growth of these cell populations in semi-solid culture systems, after incubation with cloned cytotoxic effector cells. Three different types of cloned effector cells were investigated: TCR-/CD3- NK cells, TCR-gamma delta+/CD3+ cells, and TCR-alpha beta+/CD3+ cytotoxic T lymphocytes. Effector cells showed differential levels of tumor cell colony inhibition, but no MHC-non-restricted lysis of normal HPC was observed. Pre-stimulation of normal HPC by culturing on established stromal layers had no effect. Cell-mediated lysis of HPC only occurred by Ag-specific MHC-restricted lysis by CTL, or by antibody-dependent cellular cytotoxicity. In cell mixing experiments, irradiated tumor cells, but not normal bone marrow cells inhibited tumor cell lysis. Furthermore, cloned effector lymphocytes were able to specifically eliminate malignant cells from tumor contaminated bone marrow without damaging normal HPC. When fresh leukemic cells were used as targets, growth of acute myeloblastic leukemia colonies was inhibited after incubation with several cytotoxic effector clones, whereas chronic myeloid leukemia precursor cells showed limited sensitivity to MHC-non-restricted cytolysis. These results indicate that MHC-non-restricted cytolysis by NK cells is selectively directed against neoplastic cells and not against normal HPC.     

Induction and blocking of cytolysis in CD2+, CD3- NK and CD2+, CD3+ cytotoxic T lymphocytes via CD2 50 KD sheep erythrocyte receptor

The 50 KD sheep red blood cell antigen receptor CD2 is the earliest T cell differentiation marker and is present on all blood-derived T cells, including natural killer (NK) cells. The CD2 antigen is also known to serve as an important activation site regulating various T cell functions. We report that anti-CD2 monoclonal antibodies (MAb) block MHC-restricted class I- and class II-specific cytolysis by CD2+, CD3+ clones of the relevant target cells, irrespective of whether lysis by these clones is blocked by anti-CD3 or anti-CD8 MAb. Moreover, anti-CD2 MAb (but not anti-CD3 MAb) are able to reduce MHC-nonrestricted, nonspecific cytolysis: a) by CD2+, CD3+ clones of K562 target cells; and b) by CD2+, CD3 NK clones of K562 as well as Daudi cells. Different preparations of anti-CD2 MAb vary in their capacity to inhibit cytolysis. For cloned effector cells, the percent inhibition of lysis by CLB-T11 greater than Lyt-3 MAb, whereas with "fresh" NK cells, the lysis inhibitory ability of Lyt-3 greater than CLB-T11. The antibody-dependent cellular cytotoxicity by "fresh" and cloned NK cells is not inhibited by anti-CD2 MAb. Anti-CD2 MAb also prevent the induction of lysis by cross-linked anti-CD3 MAb, e.g., by CD2+, CD3+ cloned cloned cells against (IgG-FcR+) Daudi cells. Anti-CD2 MAb can also induce cytolysis in some, but not all, CD2+, CD3- NK clones against xenogeneic P815 mouse mastocytoma cells. Anti-CD2 MAb, in combination with lectins (PHA or Con A: pretreatment of effector cells), can also induce cytolytic activity by CD2+, CD3+ clones against Daudi cells. Our data therefore support the concept that the CD2 antigen is an important activation site regulating a wide variety of T cell functions including cytolysis. Whether ligand interaction with the CD2 antigens results in augmentation or inhibition of T cell functions may very well depend on the type of CD2 antigen-ligand interaction, e.g., cross-linked ligand-receptor interaction may, in general, enhance the various T cell functions, whereas noncross-linked ligand-receptor interactions may inhibit such functions, as we and other investigators demonstrated earlier for the CD3/Ti antigen-receptor complex activation site.     

Ligation of the lymphocyte homing receptor CD44 triggers T-helper and cytolytic functions of human T cells

We show that antibodies to the CD44 molecule trigger proliferation of human CD3+/CD4+ T-cell clones. Such effect is IL2-dependent, as shown by IL2 production induced by anti-CD44 mAb and by inhibition of cell proliferation in the presence of anti-IL2 antibodies or cyclosporin A (CsA). Moreover, anti-CD44 mAb triggered human cytolytic CD4+ and CD8+ TCR /+ clones, and V1 or V2 TCR Y/+ clones to lyse Fc-gamma-R+ P815 cells and to release granule trypsin-like esterase enzymes. Anti-CD44 mAb-triggered proliferation and cytotoxicity were blocked by the PTK-inhibitor, genestein. In addition, ligation of the CD44 molecule induced tyrosine phosphorylation of proteins identical, by molecular weight, to those phosphorylated following anti-CD3 mAb-stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21 kD protein (the phosphorylated zeta chain of the TcR molecular complex) typically observed upon anti-CD3 mAb stimulation.     

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

HIV-infected humans, but not chimpanzees, have circulating cytotoxic T lymphocytes that lyse uninfected CD4+ cells

It has been suggested that autoimmune phenomena contribute to the depletion of CD4+ T cells and the development of AIDS in HIV-1 infected humans based, in part, on observations that some HIV-1-infected humans have autoantibodies reactive with Ag expressed on uninfected CD4+ cells. In this study, 11 of 14 asymptomatic HIV-1-infected homosexuals and hemophiliacs, but none of 17 uninfected homosexuals or heterosexuals, were found to have cytotoxic lymphocytes in blood that can lyse uninfected CD4+ T cells from humans and chimpanzees but not human B lymphoblastoid cells or mouse T cells. The cytotoxic PBL were concluded to be CTL rather than NK cells, with the phenotype being CD3+, TCR-1 alpha beta+, CD8+, CD4-, CD16- based on findings that PBL-mediated lysis of uninfected CD4+ cells was 1) blocked by a mAb to CD3, which inhibits CTL but not NK activity; 2) diminished by treatment of PBL with a mAb to CD8 and C, but not by treatment with mAb to CD4 or CD16 and C; and 3) blocked by mAb WT31 directed against the TCR-1 alpha beta. In contrast, PBL from HIV-1-infected chimpanzees, which to date have not developed AIDS, lacked detectable CTL lytic for uninfected CD4+ cells.     

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.     

Differential expression of CD8 alpha and CD8 beta associated with MHC-restricted and non-MHC-restricted cytolytic effector cells

The differential expression of the alpha and beta chains of the CD8 glycoprotein was examined in three functionally distinct cytolytic effector cell populations: (i) T cells (CD3+ CD56-), (ii) NK cells (CD56+ CD3-), and (iii) non-MHC-restricted T cells (CD56+ CD3+). Twenty-four percent of T cells were CD8+, and they consistently coexpressed both CD8 alpha and CD8 beta. Moreover, CD8+ T cells uniformly expressed high-density CD8 alpha. Forty percent of NK cells were CD8+ but the vast majority (approximately 75%) expressed only CD8 alpha without CD8 beta. In addition, CD8+ NK cells uniformly expressed low-density CD8 alpha. In comparison, 75% of non-MHC-restricted T lymphocytes were CD8+ but they displayed an intermediate phenotype: 60% coexpressed CD8 alpha and CD8 beta while 40% expressed only CD8 alpha. Within this population, CD8 alpha was expressed at high density, similar to that of T cells. Following IL-2 activation, enhancement of non-MHC-restricted cytotoxicity was not associated with any changes in either the quantitative or qualitative pattern of expression of CD8 alpha or CD8 beta by these cells. Addition of either anti-CD8 alpha or anti-CD8 beta mAb did not alter non-MHC-restricted cytotoxicity of either CD56+ CD3- or CD56+ CD3+ effector cells. However, within the CD56+ cell population, non-MHC-restricted cytotoxicity was almost entirely found within the CD8- and CD8 alpha + beta- populations, and both subsets displayed a similar level of killing. In contrast, CD8 alpha+ beta+ cells exhibited very little non-MHC-restricted cytotoxicity. Thus, the coexpression of CD8 alpha and CD8 beta in conjunction with the TCR/CD3 complex appears to characterize MHC restricted cells while the expression of CD8 alpha alone is associated with non-MHC-restricted cytotoxicity. Taken together, these findings suggest that neither CD8 alpha nor CD8 beta is involved in the initial phases of target cell binding or recognition during NK cell-mediated lysis. However, the selective expression of CD8 alpha by a large fraction of non-MHC-restricted effector cells suggests that this antigen may play a different functional role in this unique subset of cytolytic lymphocytes.     

Human T cell clones with gamma/delta and alpha/beta receptors are differently stimulated by monoclonal antibodies to CD2

Requirements for stimulating autocrine proliferation of human T cell clones expressing either alpha/beta or gamma/delta antigen receptors via the "alternative" CD2 pathway have been examined using a large set of monoclonal antibodies (mAb). In the presence of autologous accessory cells (AC, B-lymphoblastoid cell lines) 2 of 13 single CD2 mAb (CLB-T11.1/1 and 6F10.3) stimulated proliferation of gamma/delta but not alpha/beta cells. Interleukin (IL) 1 or IL 6 did not substitute for AC in stimulating gamma/delta clones. Addition of CD28 mAb YTH 913.12 with the CD2 mAb did not result in stimulation of any alpha/beta clones. In the absence of AC, none of the CD2 mAb singly could stimulate any T cell clones, but pairs of mAb directed to different epitopes of CD2 (CLB-T11.1/1 + CLB- T11.2/1 or 6F10.3 + 39C1.5) stimulated both alpha/beta and gamma/delta clones. In both cases, stimulation was reduced by the presence of CD3 mAb. These results confirm that the established AC-independent alternative pathway of T cell activation, which requires binding of two separate epitopes of CD2, operates in both gamma/delta and alpha/beta T cells, and further suggest that an additional pathway initiated by binding of a single CD2 epitope in the presence of AC is exclusively operational in gamma/delta cells.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.