Lectin-dependent Cytotoxicity Research Articles

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.

Human alveolar macrophages: wheat germ agglutinin-dependent tumor cell killing.

Human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors were examined for ability to cause lectin-dependent tumor cell killing. Of five plant and two animal lectins tested, only one lectin, wheat germ agglutinin (WGA), induced significant and reproducible lectin-dependent macrophage-mediated cytotoxicity (LDMC) against human bladder cancer (T-24) cells. There was no significant difference between the LDMCs of AM and blood monocytes. All 6 tumor cell lines tested were sensitive to various extents to LDMC induced by WGA. Quantitative analysis with WGA-FITC conjugate showed the presence of various levels of receptors for WGA on the surface of AM, monocytes and tumors. A relatively good correlation was found between the sensitivities of the tumor cells to LDMC mediated by AM and the numbers of receptors for WGA. Pretreatment of AM or monocytes with LPS did not affect their LDMC. These results indicate that a plant lectin, WGA which binds to both human AM and tumor cells, renders human AM cytotoxic to allogeneic tumor cells by a different mechanism(s) from that involved in the nonspecific tumor cytotoxicity of activated macrophages.

Apoptosis as a mechanism of lectin-dependent monocyte-mediated cytotoxicity.

In this study we investigated the mechanisms of cytotoxicity mediated by pokeweed mitogen (PWM)-activated human peripheral blood monocytes. By using DNA electrophoresis and propidium iodide (PI)-DNA staining flow cytometry, we demonstrated that apoptotic cell death of target U937 cells and Raji cells was induced in lectin (PWM)-dependent monocyte-mediated cytotoxicity (LDMC). The LDMC-mediated DNA fragmentation in U937 cells and Raji cells was induced in lectin (PWM)-dependent monocyte mediated cytotoxicity(LDMC). The LDMC-mediated DNA fragmentation in U937 cells was completely inhibited by anti-TNF alpha monoclonal antibody (mAb), but not by the addition of monosaccharide (N-acetylglucosamine, GlcNAc, a sugar specifically recognized by PWM and a lectin-like receptor on monocytes). In contrast, GlcNAc inhibited the DNA fragmentation in Raji cells induced by LDMC which the anti-TNF alpha mAb had no effect. PWM was found to stimulate the production of nitric oxide (NO) from monocytes. The NO-production was enhanced in the presence of target Raji cells, while the enhancement was abolished by the treatment with GlcNAc. By flow cytometry, we found that PWM bound to tumour cells as well as monocytes, and inhibited the expression of HLA-DR antigen on tumour cells. These results suggest that the presence of lectin molecules on the surface of monocytes and tumour cells may bring the two cells together, thus facilitating the induction of apoptosis in target cells by triggering the production of cytolytic factors (TNF and NO) and the modification of target cell surface antigen (HLA-DR).

Monoclonal proliferation of CD4+ large granular lymphocytes with cytolytic activity

A rare case of monoclonal proliferation of CD3+4+8- T-cell receptor-alpha beta+ large granular lymphocytes (LGL) is presented. CD4+ LGL in the present case showed spontaneous cytotoxicity against herpes simplex virus-infected cells and antibody- and lectin-dependent cytotoxicity. Perforin, which is one of the important cytolytic mediators of cytotoxic T cells (CTL) and natural killer cells, was abundantly expressed in CD4+ LGL of this case. The present case suggests that perforin-positive CD4+ CTL, which have recently been shown in the in vitro studies, certainly exist in vivo.

Selective loss of NK cytotoxicity in antisense NK-TR1 rat LGL cell lines. Abrogation of antibody-independent tumor and virus-infected target cell killing.

We have shown that NK-TR1, a protein containing a cyclophilin-like domain, is associated with a receptor/triggering molecule on the surface of human large granular lymphocytes (1). In the present study, we have further defined the role of NK-TR1 in target cell recognition/killing by generating antisense NK-TR1 transfectants in the rat LGL cell line, RNK-16. Stable transfectants were identified by analyzing permeabilized cells with the anti-NK-TR1 mAb, 4F9. Transfectants with low levels of 4F9 staining showed drastically reduced levels of killing against three NK-susceptible target cell lines. Lytic activity against vaccinia virus-infected cell lines also was dramatically reduced. In contrast, transfected cells showing normal levels of NK-TR1 expression demonstrated normal killing of all target cells. The ability of all transfectants to form conjugates was identical to that observed with the wild-type RNK cell line. Lectin-dependent cytotoxicity, reverse ADCC via NKR-PI, and ADCC-mediated killing were comparable in both high or low NK-TR1 expressing clones, demonstrating that the lytic machinery was still intact. BLT-esterase activity, PF levels, and surface marker phenotype were not significantly affected. These results provide strong evidence that NK-TR1 is an essential element in a signaling pathway leading to MHC unrestricted killing of tumor and virus-infected cells.

Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells

Purpose : To determine whether hyperthermia is to the benefit or detriment of host immune function, the effect of hyperthermia was evaluated on various functions of T-lymphocytes from human umbilical cord blood and compared to that of adult blood. Methods and Materials: Nonadherent mononuclear cells from cord blood or adult blood were used as the effector cells. To generate lymphokine activated killer (LAK) cells, effector cells were kept in culture for 5 days in complete medium containing recombinant human interleukin-2. To activate effector cells to become cytotoxic, cells were kept in culture in complete medium containing Con A. Cytotoxicity was determined in a standard 4-h chromium release assay using K-562 human erythroleukemic cells (in the natural killer cell activity assay) or Daudi cells (in the LAK cell activity or Lectin dependent cytotoxicity assay) as targets. For heat effects, cells in complete medium were heated at the desired temperature in a water bath for 1 h. Results: Lymphokine-activated killer cell activity, lectin-dependent cytotoxicity and T-cell proliferative capacity were not deficient in human cord blood. Cytotoxic activities of T-cells from adult blood as well as from cord blood can be enhanced at febrile range (⩽ 40°C), and were significantly decreased by exposure to 1 h at 42°C. Conclusion: The febrile responses (⩽ 40°C) to infection, in the course of malignant disease and with biological response modifiers treatment, may all be related to host defense mechanisms. Based on these observations, whole body hyperthermia (⩽ 40°C), in combination with the appropriate cytokines, may have therapeutic potential in the treatment of neonatal infections and malignancies under certain circumstances. Hyperthermia in febrile range may, therefore, confer an important immunoregulatory advantage to the host. In contrast, tumor killing therapeutic temperature (> 42°C) which inhibits host immunocompetence should probably be used only for local hyperthermia.

In vitro expansion and analysis of T lymphocyte microcultures obtained from the vaccination sites of cancer patients undergoing active specific immunization with autologous Newcastle-disease-virus-modified tumour cells.

In order to understand further the effects of Newcastle-disease-virus(NDV)-modified tumour vaccines we investigated the feasibility of isolating lymphocytes from the site of injection of patients undergoing postoperative active specific immunization (ASI) with autologous NDV-modified tumour cells. Delayed-type-hypersensitivity(DTH)-like reactions from five cancer patients were surgically removed, minced and the tissue particles were digested with collagenase and DNase. Lymphoid cells recovered were expanded in a highly efficient limiting-dilution analysis system optimized for T cell growth [Moretta et al. (1983) J Exp Med 157: 743] and lymphocyte microcultures (clonal probability > 0.8) could be grown for up to 1 year. Analysis of the microcultures for phenotype and function showed that the majority were positive for CD4 (92%) and TCR alpha beta (96%). Concanavalin-A-induced production of interleukin-2 (IL-2), IL-6, interferon gamma and tumour necrosis factor alpha was detected in more than 70% of the microcultures. Lectin-dependent cytotoxicity was only very rarely observed. The general characteristics of the microcultures obtained support the notion of a DTH-like reaction taking place at the site of tumour cell challenge. The possibility of in vitro expansion and cultivation of T lymphocytes from ASI vaccination sites should help to elucidate further the role of these cells in active specific immunization against autologous tumour cells.

In vivo and in vitro primed lymphocytes correlation of cytochemically detected BLT-specific lymphoid serine protease with cytotoxic activity

We describe the validation of a cytochemical method to detect a cytolytic cell-specific lymphoid serine protease which can be upregulated during viral infection and allogeneic stimulation. The cytolytic cell specificity was ascertained by demonstrating a high correlation between BLT substrate-specific serine protease (SP) activity and cytotoxicity of in vivo and in vitro stimulated lymphocytes. The presence of SP in peripheral blood lymphocytes was compared with their capacity to kill K562 targets in a lectin-dependent cytotoxicity assay. The correlation coefficient was 0.92 and 0.93 at E: T ratios 1o:1 and 20:1 respectively. In allogeneic mixed lymphocyte cultures an increase of SP activity in effector lymphocytes was paralleled by an augmentation of cytotoxic capacity towards stimulator target cells. SP + granules showed intracellular polarization to the effector/target cell interface during conjugate formation. These results together with previous studies suggest that this method provides a sensitive assay which predicts the cytolytic potential present in a lymphocyte population.

Coculture with autologous myotubes of cytotoxic T cells isolated from muscle in inflammatory myopathies

T-cell lines were expanded from muscle of 10 patients with polymyositis, 5 with inclusion body myositis, 5 with dermatomyositis, and 5 with other muscle diseases. All cell lines uniformly expressed T-cell antigens, but not natural killer cell or B-cell antigens. The proportion of helper (CD4+) and cytotoxic (CD8+) T cells in the expanded lines was variable and showed no correlation with the diagnosis. Sixteen cell lines (6 polymyositis, 4 inclusion body myositis, 5 dermatomyositis, 1 other muscle disease) consisted predominantly of CD8+ T cells. None of these lines displayed natural killer-like cytotoxicity but all were capable of lectin-dependent cytotoxicity. Three of 6 polymyositis, 1 of 4 inclusion body myositis, and 1 of 5 dermatomyositis lines showed low but statistically significant cytotoxicity against autologous myotubes (6 to 27% specific 51Cr release; effector-target ratio, 20:1). The results demonstrate that functionally competent cytotoxic T cells can be expanded from muscle affected by inflammatory myopathies and are consistent with the hypothesis that some cytotoxic T cells recognize an autoantigen on myotubes. Further studies of this experimental system may define the molecular mechanism of T cell-mediated muscle fiber injury and may help to identify the relevant antigens.

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.

Characteristics of T-Lymphocytes Infiltrating Human B-Cell Lymphomas

Using a recently described technique for direct expansion of human T-lymphocytes isolated from small intestine biopsies, we have investigated the local cellular immune response in six patients with B-cell lymphomas of various subtypes. T-cell lines (TCL) were established by seeding tumor-infiltrating T-lymphocytes (T1TL) at limiting dilution in the presence of irradiated feeder cells in culture medium containing rIl-2 and phytohemagglutinin (PHA). About 1/50 T-cells gave rise to a TCL; they all were CD3+. The CD4/CD8 ratio was 3.8:1 before and after cloning. Of 45 TCLs analysed so far from one patient with B-cell lymphoma of the lung, 4 were cytotoxic as shown by their ability to exert lectin-dependent cytotoxicity against allogeneic target cells. Of these, 3 demonstrated specificity for the autologous malignant B-cells. Five TCLs lysed the NK-sensitive K562 cell line in a HLA-unrestricted manner. When tested for antigen-specific proliferative activity, 4 TCLS only responded to the autologous lymphoma cells, but 5 TCLs reacted to the autoantigenic ganglioside GM1. Southern Blot analyses did not show a clonal pattern of T-cell receptor gene rearrangement within all TITL populations. The peripheral T-lymphocytes of the lymphoma patients showed a drastically reduced response to the mitogens PHA. Concanavalin A, and pokeweed mitogen. The present report demonstrates that it is possible to analyze TITL at clonal level. This technique may be the only means of investigating the specificity of the TITL and may help us to identify the relevant tumor-associated autoantigens if tumor-induced autoimmunization is indeed one of the mechanisms that control the growth of tumors and metastases.

Characteristics of T-lymphocytes infiltrating human B-cell lymphomas.

Using a recently described technique for direct expansion of human T-lymphocytes isolated from small intestine biopsies, we have investigated the local cellular immune response in six patients with B-cell lymphomas of various subtypes. T-cell lines (TCL) were established by seeding tumor-infiltrating T-lymphocytes (T1TL) at limiting dilution in the presence of irradiated feeder cells in culture medium containing rIl-2 and phytohemagglutinin (PHA). About 1/50 T-cells gave rise to a TCL; they all were CD3+. The CD4/CD8 ratio was 3.8:1 before and after cloning. Of 45 TCLs analysed so far from one patient with B-cell lymphoma of the lung, 4 were cytotoxic as shown by their ability to exert lectin-dependent cytotoxicity against allogeneic target cells. Of these, 3 demonstrated specificity for the autologous malignant B-cells. Five TCLs lysed the NK-sensitive K562 cell line in a HLA-unrestricted manner. When tested for antigen-specific proliferative activity, 4 TCLS only responded to the autologous lymphoma cells, but 5 TCLs reacted to the autoantigenic ganglioside GM1. Southern Blot analyses did not show a clonal pattern of T-cell receptor gene rearrangement within all TITL populations. The peripheral T-lymphocytes of the lymphoma patients showed a drastically reduced response to the mitogens PHA. Concanavalin A, and pokeweed mitogen. The present report demonstrates that it is possible to analyze TITL at clonal level. This technique may be the only means of investigating the specificity of the TITL and may help us to identify the relevant tumor-associated autoantigens if tumor-induced autoimmunization is indeed one of the mechanisms that control the growth of tumors and metastases.

Propagation and characterization of lymphocytes infiltratiti livers of patients with primary biliary cirrhosis and autoimmune hepatitis

Lymphocytes were prolagated with interleukin 2 from liver tissue removed at transplantation from patients with primary biliary cirrhosis or autoimmune chronic active hepatitis. Phenotypic analysis of the cultured lymphocytes as well as the infiltrating cells in situ indicated that the culture technique did not select for a particular phenotype. Eight cultures were tested for cell-mediated lympholysis activity against a bile duct tumor line as well as a hepatocellular carcinoma line, but no specific killing was seen. In addition, no natural killer activity was detected. However, the lymphocytescultures were able to kill the targets in a lectin-dependent cytotoxicity assay, indicating their cytolytic effector activity. Preliminary studies have demonstrated that lymphocytes extracted from hepatic tissue and hilar lymph nodes from a patient with primary biliary cirrhosis proliferated in response to autologous biliary epithelial cells. These methods might be useful in studying the pathogenesis of primary biliary cirrhosis and other liver diseases with autoimmune features.

Human oligodendrocytes are susceptible to cytolysis by major histocompatibility complex class I-restricted lymphocytes

The majority of human oligodendrocytes in enriched glial cell cultures expresses class I major histocompatibility complex (MHC) antigens. We used a 51Cr release assay to study the susceptibility of oligodendrocyte-enriched glial cells to MHC-restricted and non-restricted immune-mediated cytolysis. Mitogen-activated mononuclear cells induced significant lysis in a lectin-dependent cytotoxicity assay. Mononuclear cells allo-activated in a one-way mixed lymphocyte culture with E − cells from the glial cell donor induced a significantly higher degree of oligodendrocyte cytolysis than mononuclear cells activated with E − cells bearing MHC-class I antigens discordant with the glia. Cytolysis by alloactivated unfractionated lymphocytes and by purified CD8 + lymphocytes was reduced by an anti-class I antibody (W6/32). Our findings suggest that human oligodendrocytes can be susceptible targets for MHC class I-restricted lysis.

Frequency analysis of CD4+CD8+ T cells cloned with IL-4

The coexpression of both CD4 and CD8 molecules on T cells occurs in the peripheral blood at a low frequency and can be generated transiently on CD4+ peripheral blood T cells by treatment with lectin which induces CD8 biosynthesis and cell surface expression. We have cloned T cells in a nonselective fashion from normal subjects in the presence of either IL-2, rIL-4 and IL-2, or rIL-4 and have examined the phenotypic expression of CD4 and CD8. The addition of excess rIL-4 increased the expression of CD8 on the surface of CD4+ T cell clones but did not increase CD4 expression on CD8+ T cell clones. There were three patterns of CD4 and CD8 expression observed: high density CD8 with no CD4 expression; high density CD4 with low CD8 expression; or high density CD4 with higher cell surface CD8 expression which was regulated by the presence of rIL-4. CD4+ T cell clones originally cultured in IL-2 and rIL-4 and subsequently grown in IL-2 alone exhibited decreased expression of the CD8 molecule. The increased expression of CD8 did not correlate with NK activity or lectin-dependent cytotoxicity in an antigen independent system. In addition, rIL-4 alone or in combination with IL-2 appeared to accelerate the growth curve of T cell clones as compared to IL-2 alone. These results show that IL-4 can upregulate CD8 expression on CD4+ T cell clones while not effecting CD4 expression on CD8+ T cell clones. As class I MHC is the ligand for the CD8 molecule, expression of CD8 induced by IL-4 on CD4+ T cells may allow for increased nonspecific cell to cell contact during the course of an inflammatory response.

Proliferative activity, lectin-dependent and natural cytotoxicity in blood, lymph node and spleen from patients with Hodgkin's disease

Mononuclear cells and T-lymphocytes of the blood, spleen and lymph nodes from 48 patients with Hodgkin disease (HD) and blood donors were tested in assays for lectin-dependent (LD) and natural killer (NK) cytotoxic activity. On average, peripheral blood T cell lectin-dependent cytotoxicity differs from that of the donors. However, cytotoxic activity appears to be dependent on the stage of disease; in the IY stage LD cytotoxicity was decreased 2-fold. The lectin-dependent cytotoxicity was also dependent on the histological type of disease and the lowest level (50% of the control level) was associated with the lymphoid depletion type. The cytotoxic activity of T-lymphocytes from the affected areas of the patients' spleen was more marked than that of the unaffected areas. Spleen cell cytotoxicity showed no other correlations. Cytotoxicity of lymphocytes from the affected lymph nodes was drastically lower than activity of blood and spleen lymphocytes. NK activity of the patients' blood and spleen lymphocytes was twice as low as the control level (healthy donors) and did not correlate with stage and/or histological type of disease. The proliferative activity of lymphocytes from 33 HD patients was tested in vitro using allogeneic mononuclear cells from healthy donors or HD patients and/or PHA as stimulators. The response of patients' lymphocytes to alloantigens appeared to be much less affected than response to polyclonal mitogen. Thus, the results obtained by us demonstrate signs of stimulation of the lymphoid system against a background of general immunosuppression in HD.

EVIDENCE OF INTRAGRAFT INTERLEUKIN-2-ACTIVATED KILLER CELLS AND ALLOSPECIFIC CYTOLYTIC T LYMPHOCYTES IN REJECTING LUNG ALLOGRAFTS

In vivo cell-mediated effector mechanisms of allograft destruction were investigated in a canine single-lung transplantation model. This large animal model permits direct longitudinal studies of immune effector cells from the grafts of individual recipients by bronchoalveolar lavage (BAL). Evidence was obtained that two types of cytolytic lymphocytes act as effectors of allograft destruction. Typical allospecific cytolytic T lymphocytes, were detected late in the course of rejection in nonimmunosuppressed recipients and in cyclosporine-treated recipients during the latter stage of drug tapering. The other type of intragraft cytolytic lymphocyte was observed in the early stages of CsA dose tapering and was characterized by ability to lyse xenogeneic targets in a lectin-dependent cytotoxicity assay but inability to kill allogeneic target cells from the lung donor. These cytolytic cells were also detected in the initial stage of lung rejection in non immunosuppressed recipients and in the early period (3 days) of mixed lymphocyte culture. Current interpretation of these data is that these latter effector cells have the characteristics of IL-2-activated killer cells (IAK). Substantial delays in the detection of intragraft donor-specific CTL relative to IAK activity were observed in recipients undergoing CsA dose tapering compared with nonimmunosuppressed recipients. This finding suggests that appropriate CsA treatment may lead to prolonged inhibition of the generation of donor-specific CTL compared with induction of IAK activity. Delayed detection of intragraft donor-specific CTL paralleled the absence of such activity in donor-specific MLC of tolerant lung allografter recipients. The result of CsA therapy may, therefore, be characterized as a state of "partial unresponsiveness," since certain pathways of immune effector activity remain intact after termination of treatment. The differential effect of CsA on various pathways of allograft destruction may have important implications regarding concepts of alloreactivity and T cell-mediated immune responses.

Association of Activated Cytolytic Lung Lymphocytes with Response to Prednisone Therapy in Patients with Idiopathic Pulmonary Fibrosis

Previous studies have suggested that immunologic mechanisms may contribute to pathogeneic reactions in certain interstitial lung diseases. Cytolytic lymphocytes are major effector cells of the immune response that have not been extensively studied in these disorders. To investigate the role of activated cytolytic lymphocytes in IPF, we studied B-cell and monocyte/macrophage-depleted lymphocyte preparations isolated from BAL fluid and peripheral blood of patients with this disease (n = 10) and used a lectin-dependent cytotoxicity assay to detect activated cytolytic lymphocytes. In longitudinal studies, those patients who had cytolytic activity of BAL fluid lymphocytes (range, 8 to 35 percent, n = 4) showed significant improvement in pulmonary function (mean increase in diffusing capacity, 30 +/- 2 percent) in association with decreased BAL fluid cytolytic lymphocyte activity after prednisone treatment. In contrast, patients who initially lacked cytolytic activity in BAL fluid (n = 6) did not improve with prednisone. Activated cytolytic lymphocytes were not observed in the BAL fluid of healthy subjects. These investigations suggest a causal relationship between activated cytolytic lymphocytes in the lung and disease activity in IPF and that assays of activated cytolytic lymphocytes are helpful in identifying patients who will improve with immunosuppressive therapy.

Virus-specific and autoreactive T cell lines isolated from cerebrospinal fluid of a patient with chronic rubella panencephalitis

Using a recently described technique for expanding of human T lymphocyte populations from cerebrospinal fluid (CSF), we investigated the local cellular immune response in a patient with chronic rubella panencephalitis. A total of 328 T cell lines (TCLs) was established by seeding CSF cells at limitin dilution into histoplates in the presence of irradiated feeder cells and phytohemagglutinin (PHA)-containing conditioned medium. 80% of TCLs expressed the CD4 + CD8 −, 5% the CD4 − CD8 + phenotype and 15% of TCLs contained different proportions of CD4 + and CD8 + cells. Of 191 TCLs analyzed, 85 were cytotoxic, as shown by their lectin-dependent cytotoxicity against allogeneic uninfected target cells. Eight of them demonstrated specificity for the autologous, rubella virus-infected target cells. When testes for antigen-specific proliferative activity, 26 TCLs responded to rubella antigen, 16 TCLs reacted to myelin basic protein (MBP), four TCLs to proteolipid protein (PLP), four to galactocerebrosides and two to actin. Fourteen out of 16 MBP-specific TCLs also responded, to a minor degree, to rubella antigen and/or actin. The results showed that the persisting rubella infection had given rise to autoreactive T cells. Virus-induced autoreactivity to brain antigens may be an important pathogenic mechanism in other chronic inflammatory disorders of the CNS.

Cytokines alter target cell susceptibility to lysis. II. Evaluation of tumor infiltrating lymphocytes.

We studied the susceptibility of autologous and allogeneic tumors to lysis by human tumor infiltrating lymphocytes (TIL) after pre-incubation of the tumors with human rIFN-gamma and human rTNF-alpha. Preincubation of the tumor lines with IFN-gamma or TNF enhanced susceptibility to lysis significantly; the combination of both cytokines was more effective than either alone. Pretreatment for at least 24 h was required to enhance lytic susceptibility and maximal lysis was observed after pretreatment for 48 to 72 h. Highly specific TIL lysed only their autologous tumor targets and failed to lyse cytokine pretreated allogeneic tumor cells. In TIL populations with varying specificity, cytokine pretreatment of targets enhanced autologous lysis as well as allogeneic lysis. This cytokine-mediated effect could also be observed in a lectin-dependent cytotoxicity assay and did not correlate directly with enhanced expression of MHC class I Ag or the adhesion molecules LFA-3 and ICAM-1. These results suggest that enhancement of lysis may occur at a postbinding stage by making the target cell more sensitive to the cytotoxic factors delivered by the killer cell. The fact that lysis of cytokine treated targets by cells with LAK activity was not enhanced suggests that cells with lymphokine-activated killer activity and tumor-derived T cells kill tumor targets via different mechanisms.