Allogeneic Target Cells Research Articles

Studies Evaluating the Antitumor Activity and Toxicity of Interleukin-15, a New T Cell Growth Factor: Comparison with Interleukin-2

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34+ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN-gamma, increased TNF-beta expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34+ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34+ cells showed markedly increased percentages of Fas receptor-expressing CD34+ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients' BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34+ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN-gamma and/or TNF-alpha and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.

Cd8+ lymphocytes that kill allogeneic and xenogeneic major histocompatibility complex class I targets

CD8+ CTLs generated in a two-way MLR should lyse target cells only if these targets share a class I MHC allele with the original stimulators. Using cattle PBMCs in a two-way MLR, we generated CDS + CTLs that kill allogeneic and xenogeneic cell lines. We have named these cells MLK cells. PBMCs isolated from two unrelated animals were cultured together. After 14 days microfluorimetry analysis was performed on the MLK cells with results showing >90% CD8+ cells. RFLP analysis revealed these cells to be predominately of one animal. MLK cells were then used as effector cells in cytotoxicity assays with syngeneic, allogeneic, and xenogeneic target cells. MLK cells were able to kill all targets. Incubating MLK cells with mAbs to CD8 markedly reduced killing, suggesting a TCR-mediated cytolytic pathway. Effective cytolysis of these targets by MLK cells was dependent on class I expression. MHC class I expression-impaired mutants of allogeneic and xenogeneic tagets were not susceptible to cytolysis. Comparisons to LAK cells revealed similarities in phenotype and function to the NK1.1 — , CD8 + subset.

Differential Effects of Ifosfamide on the Capacity of Cytotoxic T Lymphocytes and Natural Killer Cells to Lyse Their Target Cells Correlate With Intracellular Glutathione Levels

We established an in vitro model to study the influence of ifosfamide treatment on intracellular glutathione (GSH) levels in activated human effector cells with specific phenotypes and immunologic functions. Besides its role as the major intracellular reductant, GSH has been shown to affect the initiation and progression of lymphocyte activation after stimulation with lectins. An incubation of activated human peripheral blood lymphocytes (PBL) with 4-hydroxyifosfamide, the activated form of ifosfamide (4-OH-IF), resulted in a depletion of the intracellular GSH levels and a significant inhibition of the proliferative capacity in a dose-dependent manner. The cytotoxic activity of separated CD3- natural killer (NK) cells and CD3+ allospecific, cytotoxic T lymphocytes (CTL), either untreated or treated with 4-OH-IF at different concentrations, was compared in a standard 51chromium release assay (CML). There were three major findings. (1) The capacity of CD3+ major histocompatibility complex (MHC)-restricted CTL to lyse their specific allogeneic target cells was substantially reduced by preincubation of the effector cells with 4-OH-IF. This inhibition of the lytic activity in CD3+ CTL correlated with a substantial depletion of the intracellular GSH levels in this population. Rapid reconstitution of depleted GSH levels and restoration of cytotoxic activity of CTL was achieved by incubation of the effector cells with thiols, eg, glutathione ester (GSH-ester) or 2-mercaptoethanesulfonate (mesna). (2) In contrast, the lytic activity in CD3- NK cells was not substantially affected (up to 100 mumol/L 4-OH-IF). This result correlates with the capacity of NK cells to maintain their intracellular GSH levels after an ifosfamide treatment. (3) In comparison with CD3+ CTL, CD3- NK cells are more resistant to an ifosfamide treatment because they have higher initial GSH levels and a more than fourfold higher relative rate of GSH synthesis.

Coexpression of two functionally independent p58 inhibitory receptors in human natural killer cell clones results in the inability to kill all normal allogeneic target cells.

In the present study, we define a group of natural killer (NK) clones (group 0) that fails to lyse all of the normal allogeneic target cells analyzed. Their specificity for HLA class I molecules was suggested by their ability to lyse class I-negative target cells and by the fact that they could lyse resistant target cells in the presence of selected anti-class I monoclonal antibodies. The use of appropriate target cells represented by either HLA-homozygous cell lines or cell transfectants revealed that these clones recognized all the HLA-C alleles. By the use of monoclonal antibodies directed to either GL183 or EB6 molecules, we showed that the EB6 molecules were responsible for the recognition of Cw4 and related alleles, while the GL183 molecules recognized Cw3 (and related C alleles). These data suggest that the GL183 and the EB6 molecules can function, in individual NK clones, as independent receptors for two different groups of HLA-C alleles, (which include all known alleles for locus C), thus resulting in their inability to lyse all normal HLA-C+ target cells. Indirect immunofluorescence and fluorescence-activated cell sorting analysis revealed that the presently defined GL183+EB6+ group 0 NK clones brightly express EB6 molecules (EB6bright) while the GL183+EB6+ group 2 clones (unable to recognize Cw4) express an EB6dull phenotype. These data also imply that the density of EB6 receptors may be critical for the generation of an optimal negative signal upon interaction with appropriate HLA-C alleles.

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation, re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway.

CD8+ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.

Induction of CD4+ and CD8+ T cell responses in efferent lymph responding to Toxoplasma gondii infection: analysis of phenotype and function

The kinetics of induction of T cell responses were examined in efferent lymph from a node draining the site of a primary inoculation of Toxoplasma gondii. The numbers of T cells increased after infection, due initially to an expansion of the CD4+ T cell population followed by an increase in the number of CD8+ T cells which coincided with the peak lymphoblast response. Proliferative responses of CD4+ T cells to T. gondii antigen were detectable from day six after infection and immune efferent lymph cells inhibited the intracellular multiplication of T. gondii in vitro. Optimum inhibition was achieved using CD8+ T cells restimulated in vitro, and the effector function appeared to be directed preferentially against the autologous rather than the allogeneic infected target cell. The results provide unique information on the induction of immune responses to T. gondii in vivo and provide evidence that both CD4+ and CD8+ T cells are necessary for the development of protective immunity induced by the S48 strain of T. gondii which is used as a live vaccine in sheep.

THE INDIRECT PATHWAY OF ALLORECOGNITION

There is evidence that T cells can "directly" recognize intact allo-MHC molecules on the surface of allogeneic stimulator or target cells, and/or "indirectly" recognize processed allo-MHC peptides presented by self antigen-presenting cells (APCs). We and others have recently demonstrated that in vivo-primed rat CD4+ T cells recognize and proliferate to specific polymorphic amino acid sequences when presented as MHC allopeptides by self APCs. Studies on the mechanisms of indirect T cell recognition of alloantigen are now reported. First, we studied the immunogenicity of 4 synthetic polymorphic class II MHC allopeptides representing full-length sequences of the hypervariable domains of RT1.Du beta (DR or I-E-like) in several responder strains: LEW (RT1(l)), ACI (RT1a), BUF (RT1b), BN (RT1n), and control syngeneic WF (RT1u) strains. Immunogenicity of the individual 25mer allopeptides varied in the different responder strains, indicating that self-restricted T cell recognition of allo-MHC peptides is determined not only by polymorphisms, but also by the responder MHC genotype. Self-restricted CD4+ T cell recognition of processed allo-MHC peptides has been shown to occur during acute skin and cardiac allograft rejection, and there is evidence that this pathway may play an important role in initiating and amplifying the immune response to allografts. T cells from LEW animals primed in vivo by WF (RT1u) vascularized renal allografts were capable of proliferating to the RT1.Du beta peptides as early as 3 days postengraftment, when presented by self APCs. We then tested the effects of various immunosuppressive drugs on self-restricted primed T cell proliferative response to an immunogenic MHC allopeptide in vitro. Methylprednisolone, cyclosporine, and FK506 inhibited the proliferative response of RT1.Du beta 2-primed LEW T cells in a dose-dependent fashion. In addition, a single injection of cyclosporine (25 mg/kg i.m.) to LEW recipients of WF renal allografts on the day of transplantation completely abolished the proliferative response of in vivo-primed T cells to RT1.Du beta 2, indicating the susceptibility of the indirect pathway of allorecognition to conventional immunosuppressive drugs.

Interleukin 1 β synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.

Lysis of allogeneic and syngeneic target cells by primary cytotoxic T lymphocytes induced in vitro by Drosophila melanogaster cells

Incubation of unprimed splenocytes directly with synthetic peptides induces CD8 + primary CTL in vitro. The ability of peptide-pretreated LPS blasts or H-2D b-expressing Drosophila melanogaster cells to induce peptide-reactive primary CTL was analysed using synthetic peptides corresponding to distinct and overlapping amino acids in SV40 T antigen D b-restricted T-cell epitopes I, II–III, and V. Peptide-pretreated, but not untreated, LPS blasts induced strong peptide-reactive CTL in splenocytes of naive C57 BL/6 mice. The reactivity of these CTL was indistinguishable from that of CTL induced by direct stimulation of spleen cells with peptides. In contrast, effector cells induced by untreated or peptide-pretreated drosophila cells expressed equivalent cytolysis both against untreated and peptide-pretreated syngeneic target cells. They also strongly lysed the allogeneic (H-2 d) P815 target cells. The cytolytic activity was expressed by CD8 + T lymphocytes. However, the lack of specificity and restriction was probably due to stimulation of LAK cells as well. The CTL-inducing potential of the drosophila cells was stable at 46°C. Peptide-activated splenocytes cultured for 1 month in medium supplemented with spent medium from drosophila growth culture were 75% B cells and 25% CD4 + T cells. Taken together, these results indicated that LPS blasts are efficient APC for induction of primary CTL and that drosophila cell-derived molecule(s) are capable of inducing broad-reactive effector cells and may also assume the function of leukocyte growth factor(s).

Proliferation, phenotype, and cytotoxicity of human lymphocytes isolated from lymph nodes invaded by melanoma cells.

The effects of human recombinant interleukin-4 (rIL-4) on metastatic melanoma (lymph node)-derived T lymphocytes cultured with human recombinant interleukin-2 (rIL-2) were studied. Lymphocytes isolated from melanoma-invaded lymph nodes were cultured in media with rIL-2 in the presence (MB-2,4) or absence (MB-2) or rIL-4 for up to 48 days. A majority of lymphocytes grown in both cultures were CD3+ T lymphocytes. Addition of rIL-4 to the rIL-2 culture abrogated the growth of the CD3-, CD56+ cell population [natural killer (NK) cell], which were present in culture with rIL-2 alone. Lymph-node-derived T lymphocytes that had expanded under MB-2,4 conditions lysed only autologous melanoma cells and they maintained the autologous-specific cytolytic activity during the entire culture period. They did not lyse K562, Daudi, or allogeneic target cells. Monoclonal antibodies (MAbs) against CD3 molecules and MHC class I molecules but not MHC class II molecules inhibited the specific lytic functions of T lymphocytes under MB-2,4 culture conditions. Collectively, these data indicate that in lymphocyte culture derived from melanoma-invaded lymph nodes, rIL-4 inhibits the rIL-2-dependent proliferation of NK cells and antigen nonspecific killer T lymphocytes and also effectively abrogates the rIL-2-induced NK and lymphokine-activated killer (LAK) activities.

Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice.

Perforin-deficient mice have been generated by homologous recombination to determine whether the effects of CD8+ cytolytic T cells and natural killer cells are mediated by pore formation involving perforin. These mice are viable and fertile and have normal numbers of CD8+ T cells and natural killer cells which do not lyse virus-infected or allogeneic fibroblasts or natural killer target cells in vitro. The mice fail to clear lymphocytic choriomeningitis virus and they eliminate fibrosarcoma tumour cells with reduced efficiency. Perforin is therefore a key effector molecule for T-cell- and natural killer-cell-mediated cytolysis.

Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among 11 different cell lines tested, two H-2d macrophage tumour lines (P388D1, RAW 264.7), an H-2d hybridoma (Sp2/0), an H-2KkDd neuroblastoma (Neuro 2a), and H-2k fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effectors that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5 h 51Cr release assay. These anti-JEV effectors recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2+ T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.

Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells

We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B−/− mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B−/− animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B−/− CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely resuced with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.

T cell receptor recognition of MHC class II alloantigens is highly cell type dependent.

According to some models of T cell education, tolerance, and autoimmunity, recognition of MHC molecules by T cells may depend on the nature of the APC expressing the MHC/Ag complex. To examine this, a panel of 23 I-Ad-restricted, alloreactive T cells were used to probe MHC class II molecules expressed on established lines representing different lineages. Surprisingly, we observed cell type-specific reactivity in the majority of the T cells. In all, 18 different reactivity patterns were identified. The patterns observed suggests that MHC reactivity can be organized into a hierarchical pattern for both the APC and the T cells. Experiments assessing T cells' avidities for allogeneic targets, ability to produce lymphokines, and expression of accessory molecules revealed no predictive correlation with the hierarchical reactivity pattern, nor did experiments measuring allogeneic target cells' expression of known accessory molecules and ability to stimulate Ag-specific hybridomas. These results suggest that the differential reactivity cannot be accounted for by accessory molecule discrepancies among the APC, but rather might reflect deficiencies in the ability of the various APC to engage the Ag-specific T cell receptor. Collectively, these data indicate that a significant fraction of allorecognition of MHC class II is cell-type dependent and that cell-type-specific recognition may relate to peptide-specific recognition requirements by the Ag receptor of alloreactive T cells.

Induction of tumour cell lysis by a bispecific antibody recognising epidermal growth factor receptor (EGFR) and CD3

A bispecific antibody construct (bAb) recognising CD3 and epidermal growth factor receptor (EGFR) was studied in vitro. Human peripheral blood lymphocytes (PBL), pre-activated with monoclonal antibody OKT-3 or with irradiated tumour cells, were armed with the bAb construct and targeted to autologous and allogeneic tumour target cells in culture. bAb EGFR×CD3 promoted significant cytolysis even at a concentration of 1 ng/ml. The specificity of target cell lysis was provided by the EGFR specificity of the bAb, as tumour cells negative for EGFR were not lysed. However, not only EGFR-positive tumour cells but also EGFR-positive normal cells were killed. Human renal cancer cell lines and the normal autologous kidney cell cultures expressing the same level of EGFR molecules were lysed to a similar extent. These results may contribute toward the planning of future clinical trials with such bAb.

CD8+ cytotoxic T lymphocytes against antigenic variants of caprine arthritis-encephalitis virus.

Cytotoxic T lymphocytes (CTL) specific for caprine arthritis-encephalitis virus (CAEV) were characterized using a colorimetric immunocytochemistry assay to measure surviving target cells. Peripheral blood lymphocytes from a CAEV-infected goat were cytotoxic to autologous, CAEV-infected dermal fibroblast target cells following in vitro stimulation of the lymphocytes with CAEV antigen. The lymphocytes were not cytotoxic to infected allogeneic target cells or to mock-infected autologous or allogeneic target cells. This CAEV antigen-specific, major histocompatibility complex-restricted cytotoxicity was mediated by CD8+ lymphocytes as demonstrated by selective depletion with anti-CD8 antibody and complement. CTL primed with one isolate of CAEV (CAEV-Co) had no detectable activity against target cells infected with either of two neutralization variants and diminished activity against target cells infected with three other neutralization variants. This apparent variability of CTL-sensitive epitopes among CAEV isolates may contribute to CAEV escaping immune control and may complicate vaccine design.

Specific and effective T-cell recognition of cells transfected with a truncated human mucin cDNA.

Epithelial cell mucin has been characterized as a tumor-specific antigen in patients with pancreatic and breast cancer. Mucins are high molecular weight glycoproteins consisting of a heavily glycosylated tandemly repeating 20-amino acid sequence. Aberrant glycosylation of mucins on carcinomatous epithelial cells leads to the exposure of novel core epitopes that are recognized by cytotoxic T lymphocytes (CTLs). We previously reported the establishment of mucin-specific CTL clones that recognize mucin expressed on the surface of EBV-immortalized B cells transfected with the mucin cDNA (MUC1). This recognition was characterized as major histocompatibility complex (MHC)-unrestricted, because of the multivalent nature of mucin. The transfectants had to be incubated with an inhibitor of O-linked glycosylation, phenyl-N-acetyl-alpha-galactosaminide (phenyl-GalNAc) in order to unmask the tandem repeat core epitope recognized by CTLs. In the present study, we examined whether mucin molecules with fewer tandem repeats are capable of MHC-unrestricted recognition by mucin-specific CTL clones. A mucin cDNA expression vector expressing a "truncated" mucin molecule that contains only two tandem repeats was constructed. We found that mucin-specific CTL clones recognize the "truncated" mucin on allogeneic target cells, showing that recognition in this case was MHC-unrestricted as well. In addition, CTL clones lysed "truncated" mucin transfectants significantly better than full-length mucin transfectants treated with phenyl-GalNAc, and controls. The "truncated" construct may represent an effective means of immunizing patients with breast and pancreatic cancer, enabling them to mount a strong and efficient immune response against mucin-bearing tumor cells.

Lysis of major histocompatibility complex class I-bearing cells in Borna disease virus-induced degenerative encephalopathy.

CD8+ as well as CD4+ T cells and macrophages are of crucial importance for the pathogenesis of Borna disease in rats. This virus-induced immunopathological disease of the brain is characterized by neurological symptoms in the acute phase and chronic debility associated with severe loss of brain tissue in the late stage. We demonstrate here the cytotoxic activity of T lymphocytes in the brain of intracerebrally infected rats. T cells isolated from the brain of infected rats lyse major histocompatibility complex (MHC) class I-bearing target cells in the absence of MHC class II. Borna disease virus (BDV)-infected syngeneic skin cells and astrocytes, the latter one of the relevant target cells in vivo, were significantly lysed whereas infected allogeneic target cells were not. Most relevant to the in vivo situation, primary brain cell cultures propagated from the hippocampus of BDV-infected rats containing considerable numbers of neurons were lysed in vitro. Blocking experiments using antibodies directed against MHC class I antigen provided further evidence for the presence and activity of classical cytotoxic T lymphocytes. Antibodies against MHC class II antigen did not influence lysis of skin target cells but had an effect on lysis of astrocytes at late time points. Lymphocytes isolated from spleen, peripheral blood, or lymph nodes did not show cytotoxic activity. These results verify, on the cellular level, earlier findings that strongly suggest the involvement of CD8+ T cells in brain cell lesions, resulting in brain atrophy long after infection of rats with BDV. This is further evidenced by the presence of CD8+ T cells in direct proximity to neuronal cell lesions. Interestingly, the cytolytic capacity, demonstrated in vitro and strongly correlated to organ destruction, does not result in elimination of the virus but the virus persists in the central nervous system.

Inhibition of normal natural killer cytotoxicity by sera from hemophilic patients

In this study we searched for circulating antibodies or other serum factors that could account for the natural killer (NK) defect observed in hemophiliacs (He) infected with the human immunodeficiency virus (HIV). We analyzed the effect of negative or positive sera for HIV from He on normal NK activity. We showed that sera from He interfered with normal NK cytotoxicity. The inhibitory activity was higher in HIV + sera and increased as the HIV disease progressed. HIV − sera also inhibited NK function, although to a lesser extent than HIV +, and it was probably due to isoimmunization through replacement treatment with plasma-derived concentrates. For each individual, no direct correlation was found between NK inhibition (NK-INH) of sera and the NK activity of He peripheral blood mononuclear cells (PBMC). Furthermore, He serum was poorly inhibitory on autologous PBMC. Preincubation of allogenic effector or target cells with He sera revealed that the inhibitory effect was the result of the reaction with these cells. A positive correlation was found by comparing NK-INH of whole He sera with the serum levels of circulating immune complexes. When the NK-INH assay was performed using the same concentration of DEAE-purified IgG from N, HIV − or HIV +, we found that HIV + AIDS IgG was more inhibitory than the others.