Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt's lymphoma cells depending on viral latency program

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitt's lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G(1) phase. However, resveratrol strongly induced apoptosis in EBV(-) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined.     

Stimulation with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines generates HLA class I-specific CTLs with different target cell avidity.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL) are potent inducers of cytotoxic T-lymphocytes (CTL) in allogeneic mixed lymphocyte cultures (MLC). The contribution of EBV antigens to the induction of cytotoxic responses was investigated by comparing CTL clones derived from allogeneic MLCs of lymphocytes from one EBV seropositive and one seronegative donor for their capacity to lyse paired EBV positive and negative targets. The majority of the clones showed a conventional "HLA-specific" cytotoxicity and lysed equally well HLA-matched LCLs and mitogen-induced T- or B-blasts. A minority of the clones from both donors exhibited an "LCL-selective" killing potential as they lysed poorly T- and B-blasts. The LCL-selective clones did not recognize EBV antigens because they could not discriminate between EBV negative Burkitt lymphoma (BL) lines and their in vitro EBV-converted sublines. MAbs to CD3, CD8, and MHC class I antigens blocked the lysis of LCLs by HLA-specific and LCL-selective CTLs with comparable efficiency suggesting that the two effector types express T-cell receptors of similar affinity. T-blasts were unable to inhibit the lysis of LCLs in cross competition assays. This correlated with a significantly lower expression of the cell adhesion molecules ICAM-1 and LFA-3. The results suggest that stimulation with allogeneic LCLs activates HLA class I-specific CTLs with variable target cell avidity. Only CTLs that act independently of the enhancing effect of cell adhesion molecules are able to lyse mitogen-induced T- and B-blasts.     

Differences in B cell growth phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt''s lymphoma cells.

Recently established Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell lines, carrying chromosomal translocations indicative of their malignant origin, have been monitored for their degree of in vitro progression towards a more 'lymphoblastoid' cell surface phenotype and growth pattern, and for their expression of three EBV latent gene products which are constitutively present in all virus-transformed normal lymphoblastoid cell lines (LCLs). BL cell lines which stably retained the original tumour biopsy phenotype on serial passage were all positive for the nuclear antigen EBNA 1 but did not express detectable amounts of two other 'transforming' proteins, EBNA 2 and the latent membrane protein (LMP). This novel pattern of EBV gene expression was also observed on direct analysis of BL biopsy tissue. All three viral proteins became detectable, however, in BL cell lines which had progressed towards a more LCL-like phenotype in vitro. This work establishes a link between B cell phenotype and the accompanying pattern of EBV latent gene expression, and identifies a novel type of EBV:cell interaction which may be unique to BL cells.     

Differential regulation of Epstein-Barr virus (EBV) latent gene expression in Burkitt lymphoma cells infected with a recombinant EBV strain

Epstein-Barr virus (EBV)-negative Burkitt lymphomas (BLs) can be infected in vitro with prototype EBV strains to study how the virus may affect the phenotype of tumor cells. Studies thus far have concentrated on the use of transforming B95-8 and nontransforming P3HR1 strains. Immunological and phenotypic differences between the sublines infected with these two strains were reported. The majority of these differences, if not all, can be attributed to the lack of EBNA-2 coding sequences in the P3HR1 strain. The recent development of a selectable Akata strain has opened up new possibilities for infecting epithelial and T cells as well. We infected five EBV-negative BL lines with the recombinant Akata virus. Our results indicate that the infected cell lines BL28, Ramos, and DG75 express EBNA-1, EBNA-2, and LMP1, the viral proteins associated with type III latency, and use both YUK and QUK splices. In contrast, two EBV-negative variants of Akata and Mutu when reinfected displayed restricted type I latency and expressed only EBNA-1. All clones of infected Mutu cells used the QUK splice exclusively. The usage of Qp was observed in a majority of Akata clones. Some Akata clones, however, were found to have double promoter usage (Qp and C/Wp) but at 4 months after infection did not express EBNA-2. The results demonstrate differential regulation of EBV latency in BLs with the same recombinant viral strain and suggest that the choice of latency type may be cell dependent. The restricted latency observed for infected Akata and Mutu cells indicates that a BL may opt for type I latency in the absence of immune pressure as well.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes.

Previous studies on Epstein-Barr virus (EBV)-positive B-cell lines have identified two distinct forms of virus latency. Lymphoblastoid cell lines generated by virus-induced transformation of normal B cells in vitro, express the full spectrum of six EBNAs and three latent membrane proteins (LMP1, LMP2A, and LMP2B); furthermore, these lines often contain a small fraction of cells spontaneously entering the lytic cycle. In contrast, Burkitt's lymphoma-derived cell lines retaining the tumor biopsy cell phenotype express only one of the latent proteins, the nuclear antigen EBNA1; such cells do not enter the lytic cycle spontaneously but may be induced to do so by treatment with such agents as tetradecanoyl phorbol acetate and anti-immunoglobulin. The present study set out to determine whether activation of full virus latent-gene expression was a necessary accompaniment to induction of the lytic cycle in Burkitt's lymphoma lines. Detailed analysis of Burkitt's lymphoma lines responding to anti-immunoglobulin treatment revealed three response pathways of EBV gene activation from EBNA1-positive latency. A first, rapid response pathway involves direct entry of cells into the lytic cycle without broadening of the pattern of latent gene expression; thereafter, the three "latent" LMPs are expressed as early lytic cycle antigens. A second, delayed response pathway in another cell subpopulation involves the activation of full latent gene expression and conversion to a lymphoblastoidlike cell phenotype. A third response pathway in yet another subpopulation involves the selective activation of LMPs, with no induction of the lytic cycle and with EBNA expression still restricted to EBNA1; this type of latent infection in B lymphocytes has hitherto not been described. Interestingly, the EBNA1+ LMP+ cells displayed some but not all of the phenotypic changes normally induced by LMP1 expression in a B-cell environment. These studies highlight the existence of four different types of EBV infection in B cells, including three distinct forms of latency, which we now term latency I, latency II, and latency III.     

Epstein-Barr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23.

Latent Epstein-Barr virus (EBV) infection and growth transformation of B lymphocytes is characterized by EBV nuclear and membrane protein expression (EBV nuclear antigen [EBNA] and latent membrane protein [LMP], respectively). LMP1 is known to be an oncogene in rodent fibroblasts and to induce B-lymphocyte activation and cellular adhesion molecules in the EBV-negative Burkitt's lymphoma cell line Louckes. EBNA-2 is required for EBV-induced growth transformation; it lowers rodent fibroblast serum dependence and specifically induces the B-lymphocyte activation antigen CD23 in Louckes cells. These initial observations are now extended through an expanded study of EBNA- and LMP1-induced phenotypic effects in a different EBV-negative B-lymphoma cell line, BJAB. LMP1 effects were also evaluated in the EBV-negative B-lymphoma cell line BL41 and the EBV-positive Burkitt's lymphoma cell line, Daudi (Daudi is deleted for EBNA-2 and does not express LMP). Previously described EBNA-2- and LMP1-transfected Louckes cells were studied in parallel. EBNA-2, from EBV-1 strains but not EBV-2, induced CD23 and CD21 expression in transfected BJAB cells. In contrast, EBNA-3C induced CD21 but not CD23, while no changes were evident in vector control-, EBNA-1-, or EBNA-LP-transfected clones. EBNAs did not affect CD10, CD30, CD39, CD40, CD44, or cellular adhesion molecules. LMP1 expression in all cell lines induced growth in large clumps and expression of the cellular adhesion molecules ICAM-1, LFA-1, and LFA-3 in those cell lines which constitutively express low levels. LMP1 expression induced marked homotypic adhesion in the BJAB cell line, despite the fact that there was no significant increase in the high constitutive BJAB LFA-1 and ICAM-1 levels, suggesting that LMP1 also induces an associated functional change in these molecules. LMP1 induction of these cellular adhesion molecules was also associated with increased heterotypic adhesion to T lymphocytes. The Burkitt's lymphoma marker, CALLA (CD10), was uniformly down regulated by LMP1 in all cell lines. In contrast, LMP1 induced unique profiles of B-lymphocyte activation antigens in the various cell lines. LMP1 induced CD23 and CD39 in BJAB; CD23 in Louckes; CD39 and CD40 in BL41; and CD21, CD40, and CD44 in Daudi. In BJAB, CD23 surface and mRNA expression were markedly increased by EBNA-2 and LMP1 coexpression, compared with EBNA-2 or LMP1 alone. This cooperative effect was CD23 specific, since no such effect was observed on another marker, CD21.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lysis of herpes simplex virus-infected targets: II. Nature of the effector cells

Peripheral blood lymphocytes (PBL) from patients with herpes simplex virus (HSV)-1 recurrences in the cornea only (Group I) exhibited reduced lysis of HSV-1-infected targets compared to PBL from patients with oral-facial and corneal HSV recurrences (Group II). The cytotoxic lymphocytes appeared to belong to a subpopulation of natural killer (NK-HSV) cells. Monoclonal antibodies to human lymphocyte differentiation antigens were used to define the surface phenotype of the NK-HSV cells. Most of the NK-HSV activity was mediated by lymphocytes expressing the surface markers Leu-7⁺ (HNK-1), OKT3⁺ (pan T), OKM1⁺ (myeloid and NK), Leu-2^? (cytotoxic/ suppressor T cell), and Leu-8^? (regulatory T cell). In contrast, lysis of K562 cells (NK-K562) was mediated by lymphocytes bearing the surface phenotype Leu-7⁺, OKT3^?, OKM1⁺, Leu-2^+/?, and Leu-8^?. The low level of NK-HSV activity in PBL from Group I donors appeared to be due to active suppression by suppressor T lymphocytes. Depletion of Leu-2⁺ cells from PBL of Group I donors resulted in significant augmentation of NK-HSV activity. Similar treatment of PBL from Group II donors either had no effect or slightly diminished the NK-HSV activity.     

Restricted expression of EBV latent genes and T-lymphocyte-detected membrane antigen in Burkitt''s lymphoma cells.

Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing.     

Human cytotoxic T lymphocytes (CTL) against Epstein-Barr virus (EBV) infected cells: EBV specificity and involvement of major histocompatibility complex determinants in the lysis exerted by anti-EBV CTL toward HLA-compatible and allogeneic target cells

Human peripheral blood lymphocytes (PBL), from anti-Epstein-Barr virus (EBV)-seropositive donors, were stimulated by EBV and were shown to be cytotoxic toward autologous, HLA-compatible, and fully allogeneic EBV-transformed target cells. The lysis was not due to natural killer (NK) cells since the target cells used were resistant to lysis by fresh PBL and by virus-stimulated PBL-depleted of AET-SRBC-rosetting T cells (the latter being still fully cytotoxic on K562 NK-susceptible target cells). Conversely only E-rosette-purified (T) lymphocytes killed EBV-transformed HLA-compatible and allogeneic target cells. Moreover, anti-MHC antibodies inhibited the cytotoxicity exerted by EBV-induced cytotoxic T lymphocytes (CTL) on both autologous and allogeneic target cells. Finally the lysis was EBV specific since PHA blasts were not killed and since only EBV-transformed cells could compete for lysis with the EBV-positive target cells. Efficient competition was achieved by EBV-transformed cells autologous or allogeneic to the targets, even when effector and target cells were fully allogeneic. All together, the data suggest that human anti-EBV CTL may recognize nonpolymorphic HLA determinants on the target cells in association with the virus-induced antigens.     

Combined treatment with interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha up-regulates the expression of HLA class I determinants in Burkitt lymphoma lines

Cell lines derived from Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt lymphoma (BL) have a low or defective expression of polymorphic HLA class I determinants compared to EBV-transformed lymphoblastoid cell lines (LCL) of normal B cell origin and are resistant to lysis by cytotoxic T lymphocytes (CTL) specific for the corresponding determinants (M. G. Masucci, S. Torsteinsdottir, J. Colombani, C. Brautbar, E. Klein, and G. Klein, Proc. Natl. Acad. Sci. USA 84, 4567, 1987; S. Torsteinsdottir, C. Brautbar, E. Klein, G. Klein, and M. G. Masucci, Int. J. Cancer, 41, 913, 1988). In order to investigate whether this phenotypic trait of the tumor cells can be modulated by agents known to enhance HLA class I antigen expression, pairs of LCL and BL lines were cultured in the presence of recombinant human interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Three low HLA A11 expressor EBV-negative BL lines, DG 75, BL 28, and BL 41, reacted significantly stronger with the anti-HLA A11 monoclonal antibody (Mab) AUF 5.13 after combined treatment with 500 U/ml IFN-gamma and 500 U/ml TNF-alpha. Reactivity with the AUF 5.13 and with other anti-polymorphic class I Mab's was up-regulated also in in vitro EBV-converted sublines of BL 28 and BL 41. The increment of antigen expression depended on the baseline expression in untreated cells. It was largest for the low expressor lines and decreased proportionally to the level of up-regulation induced by EBV conversion. Up-regulation of HLA A11 was accompanied by induction of sensitivity to HLA A11-specific CTLs in BL 28 and its converted subline E95A BL28 while BL 41 and E95A BL 41 remained resistant. The treatment did not affect significantly HLA A11 expression of two EBV-carrying, low HLA A11 expressor BL lines, WW-1-BL and WW-2-BL, and of the EBV-carrying BL 72 line that had a high spontaneous expression. The results suggest that the down-regulation of class I antigen expression is reversible in some but not all BL lines.     

Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

P53 inactivation is often observed in Burkitt''s lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein–Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (−) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection.     

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.     

Loss of transforming growth factor beta 1 receptors and its effects on the growth of EBV-transformed human B cells.

Transforming growth factor-beta (TGF-beta) is a potent negative regulator of normal human B cell growth mediated by exogenous signals, including IL-2 and low m.w. B cell growth factor 12 kDa (BCGF-12 kDa). In the present study, we investigated the regulatory linkage between viral or nonviral transformation of human B cells and the growth inhibitory effects of TGF-beta 1. A panel of EBV+ and EBV- B cell lines, derived either by in vitro EBV B cell transformation, or from cases of lymphoma was used to quantitate the negative growth effects of TGF-beta 1. The proliferative response of three EBV- B cell lines to rBCGF-12 kDa or serum was inhibited by low concentrations of TGF-beta 1 (0.2-0.5 ng/ml for 50% maximal effect), as measured by tritiated thymidine uptake and viable cellular recovery. In contrast, rBCGF-12 kDa or serum mediated proliferation of three EBV+ B cell lines was refractory to the growth inhibitory effects of TGF-beta 1. In an attempt to understand the mechanism(s) for this differential growth control in EBV+ and EBV- B cells, we studied the expression of TGF-beta 1, c-myc, and TGF-beta 1 receptors. No correlation was observed between the expression of TGF-beta 1 or c-myc gene and growth inhibition by TGF-beta 1 in the cell lines studied. Our results indicate that sensitivity or resistance to TGF-beta 1 correlated with the presence or absence (loss) of high affinity receptors for TGF-beta 1. EBV- B cell lines expressed levels of high affinity receptors similar to those found on activated normal B or T cells. In contrast, EBV+ B cell lines showed no detectable high affinity receptors. Chemical cross-linking studies with a bifunctional reagent, dissuccinimidyl suberate revealed a normal expression of type I (65-70 kDa), type II (85-90 kDa), and type III (280-300 kDa) TGF-beta 1 high affinity receptors on EBV- B cell lines. In contrast, EBV+ B cell lines did not express type I and type II receptors, whereas type III receptors were expressed but could not be inhibited by unlabeled TGF-beta 1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of nonspecific killer cells by interleukin 2-containing supernatants

In the absence of specific antigen stimulation, nonspecific killer cells were induced by culturing C57BL/6 lymph node or spleen cells with interleukin 2-containing supernatants. These supernatants were obtained from stimulation of either rat spleen cells with concanavalin A or a variant of the T cell lymphoma, EL4 (H-2b) with phorbol myristic acetate. The ability of the EL4 supernatant to induce nonspecific killer cells was abrogated by absorption with an interleukin 2-dependent T cell line or by concanavalin A-stimulated spleen cell blasts, but not by lipopolysaccharide-stimulated spleen cell blasts or by a non-interleukin 2-producing EL4 line. Partially purified interleukin 2 from EL4 supernatants could also support nonspecific killer cell induction. The induction of cytolytic cells by interleukin 2 is sensitive to gamma-irradiation and has a D omicron of 120 rad. The nonspecific killer cells induced are likely cytotoxic T lymphocytes; the majority of the precursor and effector cells bear the Thy-1 alloantigen marker. These nonspecific killer cells killed a broad spectrum of target cells, including concanavalin A- and lipopolysaccharide-induced splenic blasts of syngeneic or allogeneic mice, a syngeneic tumor, and a cloned allogeneic cytotoxic T cell line. The frequency of precursors for nonspecific killer cells in C57BL/6 lymph node and spleen cells are 1/7000 and 1/12,000, respectively. Clonal analyses revealed that these nonspecific killers exhibit heterogeneity with respect to their target cell specificities. The induction of nonspecific killers by interleukin 2-containing supernatants is partially dependent on nylon wool-adherent cells; in antigen-stimulated cultures the most specific killer cells were obtained from cultures in which nylon wool-nonadherent lymph node responder cells were stimulated with nylon wool-nonadherent allogeneic splenic stimulator cells that were treated with anti-Thy-1 antibody and complement. The relevance of these findings with respect to the frequencies and fine specificities of cytotoxic T lymphocytes generated in interleukin 2-supplemented cultures is discussed.     

Lymphocyte subsets differentially induce class II human leukocyte antigens on allogeneic microvascular endothelial cells

Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.