Lymphocytes from hepatic inflammatory infiltrate kill rat hepatocytes in primary culture. Comparison with peripheral blood lymphocytes

In the last few years it has become possible in the liver to isolate lymphocytes from inflammatory infiltrates and to culture them in vitro. Most of the lymphocyte clones obtained are CD 8+ cytotoxic cells, but interactions between these lymphocytes and hepatocytes in primary culture have not been analysed previously. In this study, cloned human T lymphocytes from liver biopsies and from the peripheral blood of patients with chronic hepatitis B or primary biliary cirrhosis, after phenotypical and functional characterization into CD 8+ or CD 4+ cytotoxic lymphocytes, were activated in an antigen-independent fashion by adding either anti CD 3 or anti CD 2/R-3 monoclonal antibodies to the cell suspension. The activated cells were then coincubated with rat hepatocytes in primary culture. The killing capacity of the activated lymphocytes was monitored by light and electron microscopy and by measurement of lactic dehydrogenase (LDH)-release into the culture medium. It was found that cytotoxic CD 8+, but not CD 4+ helper lymphocytes very effectively killed hepatocytes. The killing effect was dependent on the time of cocultivation and on effector-target (E/T) ratio. Total breakdown of the hepatocyte monolayer was achieved after 10-20 h coculture and at an E/T ratio of 10 to 1. As LDH-release in the culture medium reached about 80% of the total LDH-content, most of the hepatocytes were lysed by activated lymphocytes. Cytotoxic activity of clones obtained from different biopsies was comparable with that of clones from peripheral blood. Hepatocytes in primary culture seem to be very sensitive to the killing capacity of activated cytotoxic lymphocytes.     

Functional heterogeneity among herpes simplex virus-specific human CD4+ T cells.

One hundred thirteen HSV-specific CD4+ T cell clones were established from the PBL of a healthy person and their functional heterogeneity was investigated. All clones proliferated in response to stimulation with HSV in the presence of autologous APC. Among those, 48 clones showed cytotoxic activity to HSV-infected autologous EBV-transformed lymphoblastoid cell line, but not to HSV-infected autologous fibroblasts, HSV-infected allogeneic cells, or K562 cells (group 1). Five clones showed cytotoxicity against HSV-infected autologous cells as well as HSV-infected allogeneic cells and K562 cells (group 2). The cytotoxicity of these clones was found to be mediated by the direct killing but not by the "innocent bystander" killing of target cells. Sixty clones showed no cytotoxic activity, however, among these, 23 revealed HLA-unrestricted and nonspecific cytotoxicity in the presence of PHA in culture (group 3), and the remaining 37 did not show any cytotoxic activity even in the presence of PHA (group 4). The cytotoxic patterns of these clones did not change in activated and resting phases, suggesting that the difference in cytotoxic ability does not depend on cell cycles. The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR or anti-CD3 mAb to the culture, whereas these mAb had no effect on the cytotoxicity of group 2. All four groups of clones had helper activity for anti-HSV antibody production by autologous B cells. Moreover it was found that all groups of clones simultaneously produced IL-2, IL-4, and IFN-gamma after culture with APC followed by HSV Ag stimulation. The surface phenotype of all clones was uniformly CD2+, CD3+, CD4+, CD8-, CD29+, CD45RA-, but expression of Leu 8 was varied. These data therefore indicate that HSV-specific human CD4+ T cells are classified into at least four groups according to the presence and specificity of cytotoxicity, i.e., Th cells with HSV-specific and HLA-class II-restricted cytotoxicity, Th cells with HLA-unrestricted and nonspecific cytotoxicity, Th cells with lectin-dependent cytotoxicity, and Th cells without cytotoxic activity. The present finding of functional heterogeneity among virus-specific human CD4+ T cells might shed light on the pathogenesis of CD4+ T cell immunodeficiency, such as human retrovirus infections.     

Activity of CD4+ T-cell clones of type 1 and type 2 in generation of influenza virus-specific cytotoxic responses in vitro

The activity of distinct CD4+ T-helper cell (Th) clones in promoting secondary A/PR/8/34/Mt.S.(H1N1) (A/PR8) influenza virus-specific, class I-restricted cytotoxic T-lymphocyte (CTL) responses in vitro was examined. CD8+ T cells which had been purified by fluorescence-activated cell sorter from spleen cells of A/PR8-primed mice were used as responders. On their own, purified CD8+ T cells were unable to generate cytotoxic activity upon in vitro culture with A/PR8-infected stimulator cells. Significant cytotoxic activity was generated in cultures that were additionally supplemented with A/PR8-specific Th clones or cell-free supernatant from these clones. Although there were large differences among individual Th clones in this function, Th clones of type 1 (Th1) promoted, on average, significantly stronger cytotoxic responses than Th clones of type 2 (Th2). The differences in promotion of a cytotoxic response correlated with the amount of interleukin-2 (IL-2) or IL-4 secreted by individual Th clones. These two lymphokines accounted for the CTL-promoting activity of the respective Th clones, since addition of recombinant IL-2 (IL-2) or rIL-4 to Th-free cultures substituted fully for the respective Th clones. As observed with Th clones, rIL-2 was significantly more effective than rIL-4 in promoting a cytotoxic response. When used in combination, Th2 clones had an antagonistic effect on the generation of a CTL response by Th1 clones. This effect could be partially transferred with cell-free supernatant from activated Th2 clones and could be reversed by addition of excess rIL-2. Both consumption of IL-2 by Th2 and secretion of an inhibitory factor(s) appear to be involved in this phenomenon.     

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.     

Two-dimensional flow cytometric analysis of T cell subsets maintained in IL-2 medium after autologous mixed leukocyte reaction activation

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin-2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two-dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin-2, there was a progressive increase in the percentages of CD8+ Leu 15- cytotoxic T, CD4+ Leu 8- helper T, and CD3+ HLA-DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)-rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock-T cells and ammonium chloride-T cells in the phenotypic changes of T cell subsets after long-term culture in an interleukin-2 medium may suggest a regulatory role of the ammonium chloride-sensitive T cells in the AMLR.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Clonal analysis of the peripheral T cell compartment of the SCID-hu mouse.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.     

Colony formation by subpopulations of human T lymphocytes. VI. Further studies on colony phenotype, function, and cloning efficiency

Phytohemagglutinin (PHA)-induced colony formation in semisolid agar medium by human peripheral blood T lymphocytes showed an increasing cloning efficiency with decreasing numbers of cultured cells. Ninety percent of CD4+ cells (inducer/helper phenotype) and 20% of CD8+ cells (cytotoxic/suppressor phenotype) formed colonies when cultured at 10-200 cells/ml culture in the presence of sheep red blood cells (SRBC) and a source of interleukin-2 (IL-2). Probably all T-colony-forming cells, but none of the subsequent colony cells, expressed the Leu-8 antigen. The cloning efficiencies of FACS-sorted cells expressing the natural killer antigenic phenotypes Leu-7+ and CD16+ were found to be less than 1%. The costimulatory effect of red blood cells for colony formation was specific for SRBC and not observed in the presence of red cells obtained from seven other species including man. All T-lymphocyte colonies obtained from unseparated peripheral blood mononuclear cells expressed the CD25 antigen (IL-2 receptor) and colonies were always composed of either CD4+ or CD8+ cells. None of the colony cells expressed the Leu-8 or the CD16 antigens. By their specific morphology in agar culture the majority of colonies composed of CD4+ cells were easily recognized, but but approximately one-third of the CD4+ colonies could not be distinguished from colonies composed of CD8+ cells. On expansion of individual colonies in liquid subculture in the presence of interleukin-2, approximately 15% of the colonies developed natural killer (NK)-like cytotoxic activity, being capable of direct killing of K562 tumor cells. It is concluded that the present method for growing human T colonies exhibits the same cloning efficiency as the most efficient liquid culture systems. Individual T colonies are composed exclusively of T inducer/helper or T cytotoxic/suppressor cells, they are never of mixed phenotype, and they do not contain cells of natural killer phenotype. Regulatory mechanisms influencing colony formation are operating between and within the various subsets of T lymphocytes.     

Induction of cytotoxicity in human peripheral blood mononuclear cells by monoclonal antibody OKT3

It has been previously reported from this laboratory that incubation of PBMC with OKT3 generates potent cytotoxic lymphocytes that can be targeted by using antibody heteroconjugates consisting of anti-target cell antibody and OKT3. In the present study these conjugates were used to explore the kinetics of induction of cytotoxicity in PBMC and the subpopulations of lymphocytes involved. It was found that in addition to conjugate-dependent cytotoxicity, a considerable amount of conjugate-independent cytotoxicity was generated during OKT3 stimulation. Although the conjugate-dependent activity resided in the CD8+ population, the conjugate-independent cytotoxicity was found to be a function of CD4-/CD8- natural killer-like cells. Being largely CD3-, those cells were most likely activated by lymphokines produced by OKT3-stimulated CD3+ cells. They were capable of killing not only tumor cells but also autologous lymphocytes. The CD4+ cells of some donors were found to exhibit low but clearly demonstrable cytotoxicity. Induction of cytotoxicity was characterized as an early event in T cell activation, correlating with the kinetics of RNA synthesis. Cytotoxicity, interleukin 2 receptor expression, and DNA synthesis declined after 3 days of activation with OKT3, indicating the existence of as yet undefined regulatory mechanisms.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

Role of primary intrahepatic T-cell activation in the 'liver tolerance effect'

There is accumulating evidence suggesting that hepatic permeability to both naive and activated T lymphocytes may be unique among the solid organs. The possibility that the liver may act as a site of primary activation for CD8+ T lymphocytes is supported by experimental data and may contribute to some of the unique immunological properties of this organ, particularly its ability to induce antigen-specific tolerance. This review discusses the nature of the liver APC inducing primary T-cell activation within the liver: Kupffer cells, liver dendritic cells, liver sinusoidal endothelial cells and hepatocytes are favourably located to allow physical contact with circulating T lymphocytes. Here, we examine the capability of each cell type to act as APC for naive CD4+ or CD8+ T cells and to induce tolerance.     

Ability of human T lymphocytes to adhere to vascular endothelial cells and to augment endothelial permeability to macromolecules is linked to their state of post-thymic maturation

The accumulation of mononuclear cells at sites of chronic inflammation is dependent on a number of factors including localized adherence of lymphocytes to vascular endothelial cells (EC), cytokine-mediated increased adhesiveness of endothelium, chemotactic factors and endothelial permeability. The present study investigates two of the above attributes of lymphocyte-EC interaction: namely, the ability of maturationally distinct subpopulations of human T lymphocytes to adhere to vascular EC and to increase vascular endothelial permeability to macromolecules in an in vitro model. Thus, human T lymphocytes were separated into CD4+ CD8-helper/inducer, CD4- CD8+ cytotoxic/suppressor, CD29+ CD45RA- CD45RO+ memory, and CD29- CD45RA+ CD45RO- naive/virgin T subpopulations, were activated with PHA and PMA, and then examined for their adherence to EC and also for their effect on endothelial permeability. Upon activation, cells within each of the above four subpopulations exhibited increased adherence to EC. In contrast, resting CD29+ CD45RA- CD45RO+ memory T lymphocytes exhibited two to three times greater ability to adhere to EC than their CD29- CD45RA+ CD45RO- naive/virgin counterparts. Consistent with their increased adherence to EC, CD29+ CD45RO+ memory T lymphocytes, when activated, significantly increased endothelial permeability to albumin. Although activated CD45RA+ naive T lymphocytes exhibited increased adherence to EC, these cells failed to increase significantly endothelial permeability. Similar to their polyclonal counterparts, Ag-specific CD4+ CD29+ CD45RO+ T cell clones, but not their actively released mediators, also increased endothelial permeability via a noncytolytic mechanism(s). This ability of CD29+ CD45RO+ memory T lymphocytes to augment endothelial permeability may facilitate their transendothelial migration into extravascular space. These observations may provide additional insights into molecular mechanism(s) underlying pathophysiology of localized chronic inflammatory responses in general and more specifically selective accumulation of CD29+/CD45RO+ memory T lymphocytes at sites of chronic inflammation such as rheumatoid synovium.     

Characterization of alpha beta+ CD4- CD8- CTL lines isolated from mixed lymphocyte cultures of adult mouse spleen cells.

Several CD4- CD8- and CD4- CD8+ T cell lines and clones have been isolated from the same mixed lymphocyte cultures. They express the alpha beta T cell receptor, are CD3+, V beta 8- and heat stable antigen-, and exhibit highly specific cytolytic activity mapping to H2Db. However, monoclonal antibody against H2Db failed to block lysis by CD4- CD8- cytotoxic T lymphocytes (CTL). One possible explanation is that the "double negative" cytotoxic T lymphocytes may recognize H2Db in the context of other class I molecules (Qa/Tla). CD4 and CD8 could be detected on some lines cloned in high concentrations of EL4 supernatant and the cell surface expression was influenced by growth conditions. The alpha beta+ CD4- CD8- CTL described in this report are clearly different from the so-called "autoimmune double negatives" which suggests a function for them in normal immune responses.     

Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T cell responses stimulated by T lymphoblasts

The capacity of various stimulator cell types to present alloantigens or viral antigens to resting human CD8+ cytotoxic lymphocyte precursors (CLP) was analyzed in a limiting dilution culture system. Cell sorter-separated T lymphoblasts of both CD4+ and CD8+ phenotypes but not resting T cells were found to efficiently stimulate the clonal development of allogeneic CD8+ CLP. Thus, 5000 CD4+ T lymphoblasts activated as many (one out of 200 to one out of 300) allogeneic CLP as 50,000 peripheral blood mononuclear stimulator cells. This potent stimulator activity was found in CD4+ and CD8+ T lymphoblasts activated by mitogen, anti-T3 monoclonal antibody, or mixed leukocyte reactions. Cytotoxic T cells generated in this system were highly specific for HLA class I antigens. Furthermore, T lymphoblasts infected with mumps virus efficiently induced development of autologous CLP into CTL clones that were virus specific and self-HLA restricted, as shown by split-well analysis. The possible in vivo significance of antigen-presenting T lymphoblasts is discussed.     

Virally infected hepatocytes are resistant to perforin-dependent CTL effector mechanisms

Cell-mediated cytotoxicity plays an important role in the clearance of noncytopathic viruses from infected tissues. Perforin-dependent cytotoxic mechanisms have been noted to play an important role in the clearance of infections from multiple extrahepatic organs. In contrast, mice with defects in the Fas/Fas ligand (FasL)-mediated cytotoxicity pathway exhibit delayed clearance of adenovirus from the liver without apparent delay in the clearance of viral infections from extrahepatic organs. The present studies examined the role of cytotoxic effector mechanisms in intrahepatic immune responses to a replication-defective, recombinant beta-galactosidase-encoding adenovirus (AdCMV-lacZ). Delayed clearance of AdCMV-lacZ from the livers of FasL-defective B6.gld mice, but not perforin-deficient B6.pfp(-/-) mice, was noted despite no significant differences in initial hepatic CD8(+) T cell IFN-gamma or TNF responses or in activation of intrahepatic cytotoxic lymphocytes cells capable of killing AdCMV-lacZ-infected fibroblast targets. In contrast, AdCMV-lacZ-infected hepatocyte targets were far more sensitive to killing by intrahepatic cytotoxic lymphocytes from B6.pfp(-/-) than from B6.gld mice, and residual levels of virus-specific killing of hepatocyte targets by FasL-defective B6.gld CTL were blocked by TNF inhibition. These results suggest that inherent resistance of hepatocytes to cytotoxicity mediated by perforin-dependent mechanisms leaves Fas/FasL-dependent, cell-mediated cytotoxicity as the major pathway for CTL-mediated killing of virally infected hepatocytes and accounts for the more prominent role of perforin-independent anti-viral mechanisms in immune responses in the liver.     

Lysis of uninfected HIV-1 gp120-coated peripheral blood-derived T lymphocytes by monocyte-mediated antibody-dependent cellular cytotoxicity

Abstract Previous reports from our laboratory have demonstrated that peripheral blood monocytes (PBM) from HIV-1 infected individuals are de novo activated and are cytotoxic in vitro. Significant monocyte-antibody-dependent cellular cytotoxicity (ADCC) was obtained against HIV-1 inactivated CD4⁺ CEM target cells coated with HIV-1 in the presence of autologous seropositive serum. Based on these findings, we hypothesized that in HIV-seropositive individuals the monocytes may play an important role in vivo in the autodestruction of non-infected CD4⁺ T lymphocytes. The present study was designed to test this hypothesis. Monocytes from normal donors activated with M-CSF lysed CD4+ T cells (CEM) coated with gp120 sensitized by plasma from asymptomatic HIV-1+ individuals in a 8 h ⁵¹Cr release assay. ADCC cytotoxic activity varied from one individual to another and was a function of the dilution of the individual seropositive plasma used. We then used circulating CD3+ T lymphocytes as targets for ADCC following treatment with actinomycin D to facilitate the release of radioactive ⁵¹Cr. Like CEM, ADCC was obtained with CD3⁺ T cells coated with gp120 in the presence of HIV seropositive plasma and monocytes. Lysis was specific as T cells that were not coated with gp120 were not destroyed. These findings demonstrate that activated peripheral blood derived monocytes can destroy non-infected gp120-coated circulating T lymphocytes by an ADCC-mediated mechanism. Thus, these findings suggest that ADCC may be one mechanism operating in vivo for the destruction of non-infected CD4⁺ T lymphocytes.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Loss of specificity in cytolytic T lymphocyte clones obtained by limit dilution culture of Ly-2+ T cells

Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.