Recent progress of molecular immunology on inflammatory myopathy

The pathogenesis of polymyositis(PM), T-cell-mediated disease, and dermatomyositis(DM), humoral immunity-mediated disease, has not been clearly understood. Retroviruses(HIV, HTLV-I) may play a important role in some PM/DM patients. HTLV-I provirus was detected in infiltrating CD4+ lymphocytes, but not within the muscle fibers by PCR in situ hybridization. It is suggested that HTLV-I-associated PM is not due to direct, persistent infection of the muscle fiber by the virus, but to a T-cell-mediated immunological process triggered by the HTLV-I-infected cells. Inclusion body myositis(IBM) is characterized pathologically by vacuolated muscle fibers containing paired-helical filaments(PHFs) similar to Alzheimer-disease brain. Furthermore, prion protein and mRNA are abnormally accumulated in IBM muscle.     

Coronary aneurysm in Behcet''s disease. Report of a case

Polymyositis, dermatomyositis, and inclusion body myositis, although immunopathologically distinct, share 3 dominant histological features: inflammation, fibrosis, and loss of muscle fibers. Progress in molecular immunology and immunogenetics has enhanced our understanding of these cellular processes. Based on the T-cell receptor gene rearrangement, the autoinvasive CD8+ T cells in polymyositis and inclusion body myositis, but not dermatomyositis, are specifically selected and clonally expanded in situ by heretofore unknown muscle-specific autoantigens. The messenger RNA of cytokines is variably expressed, except for a persistent up-regulation of interleukin 1beta in inclusion body myositis and transforming growth factor beta in dermatomyositis. In inclusion body myositis, the interleukin 1, secreted by the chronically activated endomysial inflammatory cells, may participate in the formation of amyloid because it up-regulates beta-amyloid precursor protein (beta-APP) gene expression and beta-APP promoter and colocalizes with beta-APP within the vacuolated muscle fibers. In dermatomyositis, transforming growth factor beta is overexpressed in the perimysial connective tissue but is down-regulated after successful immunotherapy and reduction of inflammation and fibrosis. The degenerating muscle fibers express several antiapoptotic molecules, such as Bcl-2, and resist apoptosis-mediated cell death. In myositis, several of the identified molecules and adhesion receptors play a role in the process of inflammation, fibrosis, and muscle fiber loss, and could be targets for the design of semispecific therapeutic interventions.     

T-cell-rich large-B-cell lymphomas contain non-activated CD8+ cytolytic T cells, show increased tumor cell apoptosis, and have lower Bcl-2 expression than diffuse large-B-cell lymphomas

The factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8+ T cells expressing alphabeta T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1+ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B+ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, <25% tumor cells were bcl-2+, whereas 6 of 12 DLBCLs had >50% bcl-2+ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95+ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1+, granzyme B-). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed.     

Pulmonary fibrosis with predominant CD8 lymphocytic alveolitis and anti-Jo-1 antibodies

Interstitial lung disease (ILD) is a complication of polymyositis (PM) and dermatomyositis (DM). It often manifests itself in association with myositis-specific antisynthetase autoantibodies, among which anti-Jo-1 antibodies are the most commonly encountered. In contrast, ILD associated with anti-Jo-1 antibodies without muscle involvement is rare and not well characterized. We report four patients presenting with ILD associated with anti-Jo-1 antibodies. Histological findings of transbronchial biopsies disclosed a pattern consistent with nonspecific interstitial pneumonitis, a CD8+ lymphocytosis was found in bronchoalveolar lavage. Only one of these patients developed an "antisynthetase syndrome" with PM, after nearly 2 yrs of severe ILD. The clinical conditions of all four cases showed stabilization or improvement when cyclosporine was added to their immunosuppressive treatment. These cases confirm that a CD8+ lymphocytic interstitial lung disease may be the first, and sole manifestation of autoimmune disease associated with anti-Jo-1 antibodies. Furthermore, they suggest that this form of interstitial lung disease apparently has a poor response to steroids and cytotoxic drugs, but may respond to moderate doses of cyclosporine and azathioprine in addition to low doses of steroids.     

Platelet factor 4, reversible inhibitor of megakaryocytogenesis, protector of megakaryocytes during chemotherapy

Development of megakaryocyte (MK) from CD34+ cord blood (CB) cells in both plasma clot culture and liquid culture was significantly inhibited by human platelet factor 4 (PF4) and human transforming growth factor beta 1 (TGF beta 1). Inhibition of cell growth by PF4 was reversible judging from the fact that the CD34+ cells preincubated with PF4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF beta 1-pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF4 at 40 micrograms/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF4, TGF beta 1 blocked more cells in G1 phase. These results demonstrate that PF4 and TGF beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF4, unlike TGF beta 1, exerts its inhibitory effect on cell growth in a reversible and S phase-specific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.     

Timing of reproductive life stages

We have investigated lysis of cultured human glial cells by non-major histocompatibility complex (MHC)-restricted or 'promiscuous' CD(4+)-T lymphocytes, activated either under relatively long-term limiting dilution culture conditions in the presence of phytohemagglutinin (PHA) and interleukin (IL)-2, or under short-term PHA-activated bulk culture conditions. Specific effector:target cell ratio-dependent lysis of oligodendrocytes (OGCs) by CD4+ T lymphocytes, generated under both of the above conditions, was observed in an 18-h 51Cr-release assay, but not in a 5-h assay. The extent of CD4 T-cell-mediated OGC lysis was less than for the tumor necrosis factor (TNF)-alpha-sensitive cell line U937, but greater than for TNF-resistant cell lines (K562, EL4). The effect could not be reproduced by T-cell culture supernatants or by high concentrations of recombinant TNF-alpha or beta. Anti-TNF-alpha antibody did not inhibit CD4-mediated lysis of OGC or U937 cells, but did inhibit U937 lysis induced by recombinant TNF-alpha, added in amounts exceeding those secreted by CD4 T cells. Human astrocytes and microglia were also susceptible to CD4+ T-cell-mediated lysis. Our results suggest that non-antigen non-MHC-restricted CD4+ T-cell-mediated injury of human glial cells can occur and may be dependent or enhanced by effector:target cell contact.     

Association of HLA class I and class II alleles with myositis in Japanese patients

OBJECTIVE: To investigate the correlation of HLA class I and class II antigens and alleles with various forms of myositis in Japanese patients. METHODS: Eighty-four Japanese patients with myositis [22 with polymyositis (PM), 46 with dermatomyositis (DM), 16 with myositis overlapping with other collagen vascular diseases] were typed serologically for HLA-A, B, C antigens. HLA-DRB1, DQA1, and DQB1 alleles were determined by polymerase chain reaction dependent DNA typing methods. Fifty-eight Japanese controls were typed serologically while HLA-DRB1, DQA1, and DQB1 allele typing was carried out in 175, 95, and 104 controls, respectively. RESULTS: HLA-B7 was higher in patients than controls [20.2 vs 6.9% in controls: p=0.02, odds ratio (OR)=3.4]. The increase of HLA-B7 was largely dependent on the increase in overlap patients (37.5%; p=0.005, OR=8.1). HLA-A24 and B52 were significantly decreased in PM as compared to DM, while CW3 was significantly increased in PM versus DM. DRB1*08 alleles were significantly increased in patients (36.9 vs 20.5% in controls; p=0.004, OR=2.3), especially in PM and DM. DQA1*0501 and DQB1*0301 were significantly decreased in patients [4.8 vs 13.7% in controls; p=0.04, OR=0.32, and 8.3 vs 20.2% in controls; p=0.02, OR=0.36, respectively]. CONCLUSION: HLA-class I and class II alleles associated with Japanese patients with myositis may be different from those associated with Caucasian patients.     

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.     

Lack of selectin-dependent adhesion in prostate cancer cells expressing sialyl Le(x)

In the muscle lesions of inclusion body myositis (IBM), two populations of T cells can morphologically be distinguished, T cells that invade muscle fibers and T cells that remain in interstitial areas. Using combined immunohistochemistry and RT-PCR, we analysed the TCR expressed by these distinct T cell populations. In three of eight IBM muscle specimens, the autoinvasive T cells were stained with one of the available mAbs: anti-Vbeta5.3 in patient 1, anti-Vbeta5.2 in patient 2, and anti-Vbeta3 in patient 3. The corresponding TCR Vbeta mRNAs were amplified by RT-PCR and the PCR products were cloned and sequenced. In all three specimens, the TCRs expressed by the autoinvasive T cells were clonally restricted. Furthermore, in patient 1 two different mAbs labelled two distinct populations of T cells: an autoaggressive population of CD8 + Vbeta5.3 + cells that invaded muscle fibers and a noninvasive interstitial population of CD8 - Vbeta5.1 + cells. TCR sequence analysis revealed that the noninvasive Vbeta5.1 + T cells were clonally diverse, whereas the autoinvasive Vbeta5.3 + T cells were clonally restricted.     

Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis

This study was performed to determine whether peritoneal T cells are suppressed in the CD4+ or CD8+ T cell subpopulation and whether they are Th1 or Th2 predominant in women with endometriosis. Immune cells in the peritoneal fluid (PF) were obtained from women undergoing laparoscopy for endometriosis or tubal ligation. Three-colour flow cytometry was utilized for immunophenotyping of peritoneal fluid mononuclear cells (PFMC). Concentrations of interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma) produced by PFMC with and without mitogen stimulation and concentrations of IL-10 and IL-12 were measured in PF. The peritoneal T lymphocytes were predominantly of the Th1 type that produced much more IFN-gamma but less IL-4 or IL-5 in women with or without endometriosis. The decrease in peritoneal lymphocytes was significant in the HLA-DR+ CD4+ CD3+ subpopulation and the concentrations of peritoneal IL-10 and IL-12 were significantly elevated in women with early stage endometriosis. There was impaired IL-5 production by PFMC after phytohaemagglutinin stimulation in women with advanced stage endometriosis. We concluded that the activated peritoneal CD4+ Th1 cells from the women with endometriosis were decreased in number. The suppression of these T cells may be due to the elevation of IL-10 and IL-12 in the peritoneal fluid.     

Perforin-deficient CD8+ T cells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-gamma but requires TNF-alpha

CD8+ T cells are effective mediators of immunity against Listeria monocytogenes, but the mechanisms by which they provide antilisterial immunity are poorly understood. CD8+ T cells efficiently lyse target cells in vitro by at least two independent pathways. To test the hypothesis that CD8+ T cell-mediated immunity to L. monocytogenes is dependent on perforin or CD95 (Fas, Apo-1), we used C57BI/6 (B6) and perforin-deficient (PO) mice to generate CD8+ T cell lines specific for the L. mono cytogenes-encoded Ag listeriolysin O (LLO). Both lines specifically produce IFN-gamma and TNF-alpha, and mediate target cell lysis in vitro. Cytolysis mediated by the PO-derived CD8+ T cell line is delayed relative to the B6-derived line and is completely inhibited by anti-CD95 Abs. In vivo, PO-derived CD8+ T cells provide specific antilisterial immunity in B6 hosts, CD95-deficient hosts, and IFN-gamma-depleted hosts. However, PO-derived CD8+ T cells fail to provide antilisterial immunity in hosts depleted of TNF-alpha. These results indicate that single Ag-specific CD8+ T cells derived from PO mice can mediate antilisterial immunity by a mechanism that is independent of CD95 or IFN-gamma, but requires TNF-alpha.     

Concentration-dependent effects of insulin on Ca2+ influx in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats

The levels of CD26 expression, their capacity to induce protein tyrosine phosphorylation and their functional implication in natural killer (NK) cytolysis have been studied. It was found that only a small fraction (12-15%) of peripheral NK cells expresses CD26 compared with the high expression (99%) found in NK clones. The protein tyrosine phosphorylation mediated by means of CD26 activation was studied in NK cells treated with the anti-CD26 MoAb 134-2C2, and two new proteins of 50 and 21 kDa appeared phosphorylated in tyrosine residues. To study the influence of CD26 antigen in NK lysis, we analysed the lytic capacity of NK cells stimulated with different anti-CD26 MoAbs or after separation into CD26+ and CD26- subsets and using K562 as target cells. Under these conditions, no differences were found in the chromium release by the target cells. Redirected lysis through CD16 was also measured by arming the effector cells (CD26+ and CD26-) with anti-CD16 antibody and using K562 as target cells. It was found that CD26- cells showed significantly less CD16-dependent lysis than CD26+ cells. These results indicate that CD26 is related to the CD16-dependent lysis but not to NK cytolysis which may be caused by mediation of protein tyrosine phosphorylation.     

Improving tumour targeting and decreasing normal tissue uptake by optimizing the stoichiometry of a two-step biotinylated-antibody/streptavidin-based targeting strategy: studies in a nude mouse xenograft model

Peripheral T-cell lymphomas (PTCLs) are often diagnosed after demonstration of T-lineage-related antigen expression on neoplastic lymphocytes by paraffin immunoperoxidase (PIP). However, complete T-cell subset analysis for helper, suppressor/cytotoxic, alphabeta, and gammadelta phenotypes has not been examined by PIP. Therefore, PIP was performed for CD4, CD8, T-cell intracellular antigen (TIA)-1, and betaF1 expression in 31 PTCLs previously studied for CD4 and CD8 by flow cytometry. The CD4 and CD8 results from both methods were compared. All betaF1- PTCLs were studied for T-cell receptor (TCR)gammadelta by PIP. PIP showed 71% correlation with the 21 PTCLs that had distinct CD4+ CD8- or CD4- CD8+ phenotypes by flow cytometry, with 64% and 90% sensitivity for CD4 and CD8 expression, respectively. Tumor cells in four of six PTCLs that had no clear CD4 or CD8 predominance or coexpression of these antigens by flow cytometry were shown to be CD4+ CD8- or CD4- CD8+ by PIP. Twelve (39%) PTCLs demonstrated a cytotoxic (TIA-1+) phenotype by PIP, including eight CD4- CD8+, one CD4+ CD8- and three CD4- CD8- cases. Of 30 immunoreactive PTCLs, 26 (87%) were alphabeta (betaF1+) by PIP. Both large cell cases among four betaF1- PTCLs were TCRgammadelta+ by PIP, including one gammadelta+ case confirmed by flow cytometry. We conclude that CD4 and CD8 T-cell subsets can be assigned for most PTCLs by PIP, with CD4 showing moderate and CD8 showing strong correlation with flow cytometric results. PIP can also define CD4 or CD8 expression on tumor cells in the PTCLs in which flow cytometry produces inconclusive results. Cytotoxic PTCLs can be identified easily with TIA-1, which can also distinguish cytotoxic from "suppressor" CD8+ PTCLs. Most PTCLs are derived from alphabeta T-cells, however some large cell gammadelta PTCLs may be identified by PIP.     

Human purified protein derivative-specific CD4+ T cells use both CD95-dependent and CD95-independent cytolytic mechanisms

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.     

Expression of MHC and cell adhesion molecules in muscular sarcoidosis

Biopsied skeletal muscles from 5 patients with muscular sarcoidosis (nodular type; 1, and myopathic type; 4) were immunocytochemically examined. All biopsies presented granulomatous changes. Atrophic or regenerating muscle fibers adjacent to granuloma demonstrated compression or ischemic changes. In the center of the granuloma, CD68+ epitheloid cells and giant cells, and CD4+ T cells were localized. At the periphery of the granuloma, CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages were found. Expression of HLA-A,B,C was diffuse in the muscle fibers. Expression of HLA-DR and ICAM-1 was more prominent near the granuloma or perifascicular fibers, and that of LFA-3 was moderate in those lesions. VCAM-1 was expressed in endothelial cells and macrophages near the granuloma. Those findings indicate that interferon-gamma or TNF-alpha produced by infiltrating inflammatory cells may induce expression of these immunologic markers or adhesion molecules. Immunocytochemical differences between the nodular and myopathic forms of sarcoidosis are not evident, but either localization or abundance of granuloma in muscle bulks is relevant to weakness or atrophy of clinically affected muscle.     

Regulation of T lymphocyte trafficking into lymph nodes during an immune response by the chemokines macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta

By virtue of their target cell specificity, chemokines have the potential to selectively recruit leukocyte subpopulations into sites of inflammation. Their role in regulation of T lymphocyte traffic into lymph nodes during the development of an immune response has not previously been explored. The sensitization phase of contact hypersensitivity induced by the hapten, dinitrofluorobenzene (DNFB) in the mouse was used as a model of T lymphocyte trafficking in response to antigenic stimulation. Rapid accumulation of CD8+ and CD4+ T cells in the draining lymph nodes was closely associated with strongly enhanced expression of macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta mRNAs and proteins. Mast cells accumulating in the nodes during DNFB sensitization were the predominant source of MIP-1 beta, whereas MIP-1 alpha was expressed by multiple cell types. Neutralization of these chemokines profoundly inhibited T lymphocyte trafficking into lymph nodes and altered the outcome of a subsequent challenge to DNFB. Thus, beta-chemokines regulate T lymphocyte emigration from the circulation into lymph nodes during an immune response and contribute significantly to the immunologic outcome.     

Characterization of rat brain stathmin isoforms by two-dimensional gel electrophoresis-matrix assisted laser desorption/ionization and electrospray ionization-ion trap mass spectrometry

A population of human T cells expressing an invariant V alpha 24 J alpha Q T cell antigen receptor (TCR) alpha chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other CD161(+) T cells, the major histocompatibility complex-like nonpolymorphic CD1d molecule is the target for the TCR expressed by these T cells (V alpha 24(invt) T cells) and by the homologous murine NK1 (NKR-P1C)+ T cell population. In this report, CD161 was shown to act as a specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by V alpha 24(invt) T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in V alpha 24(invt) T cell activation, and costimulation through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited V alpha 24(invt) T cell proliferation and cytokine secretion in response to CD1d+ target cells, demonstrating a physiological accessory molecule function for CD161. However, CD1d-restricted target cell lysis by activated V alpha 24(invt) T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in V alpha 24(invt) T cell costimulation through CD161 did not appear to involve stable association with tyrosine kinase p56(Lck). These results demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human V alpha 24(invt) T cells.     

Immune CD8+ T lymphocytes lyse Listeria monocytogenes-infected hepatocytes by a classical MHC class I-restricted mechanism

Hepatocytes constitute the principal site of listerial replication in the livers of mice infected i.v. CD8+ T lymphocytes play a predominant role in the host defenses to Listeria monocytogenes. In vitro experiments by others undertaken to delineate the functions of CD8+ T lymphocytes have focused primarily on their interaction with Listeria-infected macrophages. Such experiments do not address directly the role of CD8+ T lymphocytes in eliminating the bulk of Listeria replicating within the liver. Here, we report that immune CD8+ T cells at an E:T cell ratio > or = 10:1 lysed Listeria-infected hepatocytes as judged by the following two criteria. Aspartate aminotransferase activity in the culture supernatants, indicative of hepatocyte damage, increased significantly. Conversely, infected hepatocytes cocultured with immune CD8+ T cells exhibited a marked reduction in viable intracellular Listeria assessed by CFUs. Neither immune CD4+ T cells nor nonimmune CD8+ T cells caused a similar increase in aspartate aminotransferase activity released or a decrease in intracellular bacteria. Immune CD8+ T cell-mediated lysis of infected hepatocytes was restricted by classical MHC class I (H-2Kb) molecules and was inhibited by the presence of either brefeldin A or mAb specific for CD8. These results suggest that the predominant role of CD8+ T lymphocytes in host resistance to listerial infections of the liver may be due to their capacity to lyse infected hepatocytes.     

CD56+ putative natural killer cell lymphomas: production of cytolytic effectors and related proteins mediating tumor cell apoptosis?

Apoptosis is a regulated form of cell death that may be triggered by natural killer (NK) or cytotoxic T cells, which effect target cell lysis by cytolytic effector and related proteins through complex intracellular signals. This study was aimed to investigate whether there is selective expression of these cytolytic markers in the putative NK-cell lymphomas and whether there is correlation with zonal tumor cell death in these tumors. Expression of the cytolytic effectors perforin, granzyme B9, and the granule membrane protein TIA1 were examined in 24 putative NK-cell lymphomas, 18 postthymic T-cell lymphomas (one case CD8+ CD56+ and three anaplastic large cell lymphomas (ALCL), three T-lymphoblastic lymphomas, and 20 B-cell lymphomas. Nineteen (79%) putative NK-cell lymphomas expressed perforin, and all 24 cases expressed granzyme B9 and TIA1. The only CD8+ CD56+ postthymic T-cell lymphoma also expressed all three cytolytic markers, two CD8- ALCL expressed TIA1; other postthymic T-cell, T-lymphoblastic, and B-cell lymphomas were consistently negative. There was strong correlation between percentage perforin-positive cells and zonal tumor cell death. Angioinvasion, in contrast, was present only in a proportion (37%) of these lymphomas despite the frequent presence of zonal tumor cell death (71%). We propose that cytolytic effector and related proteins produced by putative NK and some CD8+ CD56+ postthymic T-cell lymphomas, probably in conjunction with other mechanisms, may effect massive tumor cell apoptosis. The frequent expression of cytolytic effector markers in the CD2+ surface CD3- CD56+ putative NK-cell lymphomas lends further support to their probable NK cell origin.