Derivation of herpesvirus saimiri-transformed CD8+ T cell lines with noncytotoxic anti-HIV activity

Herpesvirus saimiri (HVS), strain 488-77, was used to derive continuously growing transformed human CD8+ T cell lines that can suppress HIV replication in CD4+ cells via the production of an antiviral factor(s). Transformed CD8+ cell lines were obtained by HVS infection of peripheral blood mononuclear cells or purified CD8- T cells from HIV-infected or uninfected individuals. Suppression of primary or laboratory isolates of HIV was mediated by factor permeation of a transwell membrane or by cell-free culture supernatants. Suppressing and nonsuppressing cell lines were IL-2-dependent for good growth and showed a similar activated cell surface phenotype. The cell lines produced varying amounts of the cytokines IL-8, IL-10, TNF-alpha, TNF-beta, RANTES, MIP-1 alpha, and MIP-1 beta, but not IFN-alpha. No correlation was observed between the level of any of these cytokines and the presence or absence of antiviral activity in cell line culture supernatants. These cell lines have become an important resource for studying antiviral factors produced by CD8+ T cells from HIV-infected individuals.     

Band shift analysis of three base-pair repeat alleles in the short tandem repeat locus D12S391

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.     

The U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 do not confer acute pathogenicity upon SIVagm

Two chimeric proviruses comprising the U3 promoter and the nef gene of simian immunodeficiency virus (SIV) smmPBj1.9 in addition to other genomic regions of SIVagm3mc from African green monkeys (Cercopithecus aethiops) were constructed. The derived chimeric viruses (SIVagm3mc/SIVsmmPBj1.9) were both able to replicate in nonstimulated peripheral blood leukocytes from pig-tailed macaques (Macaca nemestrina), a biological property often correlated with acute pathogenicity. However, only one of the chimeric viruses was acutely pathogenic, inducing a rapid depletion of the peripheral CD4+ T cells in two infected pig-tailed macaques within 10 days after infection in a manner similar to infection with SIVsmmPBj1.9 itself. The other chimeric virus actively replicated during the first 8 weeks after experimental infection of two pig-tailed macaques but induced neither acute disease nor CD4+ T-cell depletion for 113 weeks after infection. Thus, the U3 promoter and the nef gene of SIVsmmPBj1.9 alone appear to be insufficient to confer acute pathogenicity to SIVagm3mc.     

Monocyte chemotactic peptide-1 expression and monocyte infiltration in acute renal transplant rejection

Disseminated infection caused by organisms of Mycobacterium avium complex is common in acquired immune deficiency syndrome (AIDS) patients. M. avium is an intracellular bacterium that multiplies within macrophages. We examined the effect of M. avium infection on the T-helper cell response in C57/BL/6 black mice. At weekly intervals, CD4+ T-cells were isolated from spleens and lines were created. T-cell lines were exposed to sonicated M. avium in the presence of feeder cells and macrophages and the supernatant were collected to measure the concentrations of interferon-gamma (IFN-gamma and interleukin-10 (IL-10). Production of IFN-gamma in CD4+ T-cells obtained from uninfected mice did not vary significantly during the 5 weeks. Levels of IFN-gamma produced by T-cell lines of infected mice were similar to the control mice during the first 2 weeks but significantly reduced (approximately 30 ng/ml) thereafter. In contrast, production of IL-10 by T-cell lines of infected mice was in a range of 190 to 342 pg/ml in weeks 1, 2 and 3, but increased to an average of 1300 pg/ml at weeks 4 and 5. Pre-immunized mice, when infected with M. avium strain 101, showed a different profile of T-cell cytokines, with high IFN-gamma and low IL-10 production. Proteins purified from a number of disease-associated (D-A) and non-D-A strains of M. avium were tested for the ability to induce IL-10. 65,000 MW and 60,000 MW proteins of M. avium induced significantly more IL-10 than 45,000 MW, 33,000 MW and 27,000 MW proteins. These results showed that M. avium predominantly stimulates either Th1 or Th2 T-helper cells according to the phase of the infection.     

Growth transformation of antigen-specific T cell lines from rhesus monkeys by herpesvirus saimiri

This study aims in establishing the in vitro basis for a primate model to evaluate potential applications of H. saimiri-transformed T cells. T cell lines specific for myelin basic protein and streptolysin O were derived from rhesus monkeys and transformed to stable antigen-independent growth with strain C488 of H. saimiri. The transformed T cells from rhesus monkeys did not produce infectious virus and harbored the H. saimiri genome exclusively in an episomal form, whereas transformed T cells from the New World monkey Calltithrix jacchus released infectious virus. Transformed T cells from rhesus monkeys showed an unaltered surface expression of CD2 and CD3, of the activation markers CD25 and CD69, and of the costimulatory molecule CD80 (B7.1). Remarkably, both transformed and nontransformed T cell lines were largely double-positive for CD4 and CD8. In contrast to the parental cell lines, the transformed cells constitutively expressed major histocompatibility complex-DR antigens and were able to present antigen to each other. The transformed T cells from rhesus monkeys continued to express a functionally intact T cell receptor and responded to recognition of their antigen with enhanced proliferation and production of Th1-type cytokines. In conclusion, H. saimiri-transformed rhesus monkey T cells may open a way to primate models for adoptive immunotherapy and studies on the pathogenesis of autoaggressive T cells.     

Patterns of lymphokine production by Semliki Forest virus-specific T-cell hybridomas stimulated with different antigen-presenting cells

The development of infection seems to be influenced by the characteristics of antigen-presenting cells (APC) in the infection site. Thus, we compared the Semliki Forest virus (SFV)-antigen-presenting capacity of spleen cells, B-cell lymphomas, bone marrow-derived mast cells and nonparenchymal liver cells by measuring the production of lymphokines in SFV-specific T-cell hybridomas. Spleen cells were able to provide the signals needed to stimulate the production of IL-2, IL-4, IL-6 and IFN-gamma, while B lymphomas the signals leading to only IL-2 production. When bone marrow-derived mast cells were used as APC, SFV-specific T-cell hybridomas produced IL-2, IL-4 and IL-6 in the presence of soluble anti-CD3 antibody. However, no lymphokine production was detected when the SFV antigen was used instead of the antibody. Nonparenchymal liver cells containing liver endothelial cells and Kupffer cells have an APC function stimulating the production of IL-2 and IL-6. These findings confirmed that the T-cell hybridomas can be selectively stimulated by different APC to produce different lymphokines, and it would influence the development of the immune-mediated inflammatory response.     

Live attenuated simian immunodeficiency virus (SIV)mac in macaques can induce protection against mucosal infection with SIVsm

OBJECTIVE: To investigate whether vaccination of macaques with attenuated simian immunodeficiency virus (SIV)macC8 could induce long-term protective immunity against rectal exposure to SIVsm and intravenous exposure to the more divergent HIV-2. DESIGN AND METHODS: Eight months after vaccination with live attenuated SIVmacC8, four cynomolgus monkeys were challenged with SIVsm intrarectally and another four vaccinated monkeys were challenged with HIV-2 intravenously. Sixteen months after SIVmacC8 vaccination, another two monkeys were challenged with SIVsm across the rectal mucosa. Two vaccinees shown to be protected against SIVsm were rechallenged 8 months after the first challenge. Ten naive animals were used as controls. Serum antigenaemia, virus isolation, antibody responses, cell-mediated immunity and CD4+ and CD8+ T-cell subpopulations were monitored. PCR-based assays were used to distinguish between virus populations. RESULTS: At the time of challenge, eight out of 10 vaccinees were PCR-positive for SIVmacC8 DNA but no virus could be isolated from peripheral blood mononuclear cells. After SIVsm challenge, three out of six vaccinees were repeatedly SIVsm PCR-negative. In one of the three infected monkeys, the challenge virus was initially suppressed but the monkey ultimately developed AIDS after increased replication of the pathogenic virus. Rechallenged monkeys remained protected. All HIV-2-challenged vaccinees became superinfected. All controls became infected with either SIVsm or HIV-2. At the time of challenge the vaccinees had neutralizing antibodies to SIVmac but no demonstrable cross-neutralizing antibodies to SIVsm or HIV-2. Titres of antigen-binding or neutralizing antibodies did not correlate with protection. Cytotoxic T-cell responses to SIV Gag/Pol and virus-specific T-cell proliferative responses were low. CONCLUSION: The live attenuated SIVmacC8 vaccine was able to induce long-term protection against heterologous intrarectal SIVsm challenge in a proportion of macaques but not against the more divergent HIV-2, which was given intravenously.     

Human immature thymocytes as target cells of the leukemogenic activity of human T-cell leukemia virus type I

The risk of developing adult T-cell leukemia (ATL) associated with neonatal infection by human T-cell leukemia virus type I (HTLV-I) suggests that early events triggered by HTLV-I might be of crucial importance in initiating the multistep lymphoproliferative process leading several decades later to the development of leukemic disease. Thus, infection of thymocytes early in life might be directly correlated with the development of ATL. In the present study, we show that in vitro infection of mature (CD2+CD3+) or immature (CD2+CD3-) thymocytes resulted in the exogenous interleukin (IL)-2-dependent proliferation of HTLV-I-positive thymocytes, most of them displaying a CD2+CD3-CD4+ phenotype and expressing the CD25 molecule, the alpha chain of the IL-2 receptor. Furthermore, the CD80 and CD54 antigens, normally expressed by thymic stromal cells, were detected on these transformed thymocytes, indicating that HTLV-I infection may disturb the cooperation between thymocytes and their thymic environment. These HTLV-I-positive thymocytes were producing significant amounts of IL-6, which was found to be implicated in their proliferation and in the expression of CD25, as demonstrated by blocking experiments using a monoclonal antibody to IL-6. The present study suggests that immature thymocytes may provide an environment favorable to the unfolding of events leading to leukemia.     

Effects of beta2 adrenoceptor agonists on T-cell subpopulations

The aim of the present communication is to determine the effects of beta2 adrenoceptor agonists on growth and cytokine secretion using allergen-specific T cells. Four beta2 adrenoceptor agonists were administered at therapeutically relevant doses (salbutamol 1-2 microM; salmeterol 0.03-0.06 microM; terbutaline 0.56-1.12 microM, and fenoterol 0.7-1.4 microM to: a) Cultures of human peripheral mononuclear cells (PBMC) b) Positively selected CD4+ and CD8+ subsets, c) Allergen-specific T-cell lines (TCL). Drug effects on growth kinetics and the secretion of IL-4, IL-5, INF-gamma and IgE following T-cell stimulation were investigated. Comparing the growth inhibitory effect of the 4 beta2 agonists at 2 different concentrations, using 12 PBMC, 10 CD4+ and CD8+ and 10 TCL cultures, the following patterns were observed: PBMC-, CD4+- and CD8+-cultures: salmeterol, followed by salbutamol and fenoterol, was a more potent inhibitor than terbutaline. In long-term TCL-cultures, salmeterol was the most potent drug, followed by fenoterol. No significant differences were observed between salbutamol and terbutaline. TCL secretion of IL-4 and IL-5 (TH2 cytokines) was also significantly inhibited. In one patient, INF-gamma secretion (TH1/THO cytokine) could be enhanced by drug administration. High IgE secretion, from 1% remaining B cells in one of the patients, following PHA+IL-2 stimulation, could be reduced by the drugs. The results showed that the beta2 agonists could influence T-cell growth and function. The changes regarding cell function were individual and related to T-cell phenotypes secreting TH1/THO or TH2 cytokines. These results suggest that administration of beta2 adrenoceptor agonists could be beneficial, not only for bronchodilation, but also for suppressing the underlying inflammatory process dominated by TH2-like cytokine secretion.     

Functional diversity of the CD8(+) T-cell response to Epstein-Barr virus (EBV): implications for the pathogenesis of EBV-associated lymphoproliferative disorders

Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8(+) T-cell responses are not uniformly cytotoxic and that CD8(+) T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4(+) T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8(+) T cells, the first of which expressed high levels of interferon gamma (IFNgamma), but little or no interleukin-4 (IL-4), whereas the second subset was IFNgamma+/IL-4(+) double-positive. A significant proportion of EBV-specific CD8(+) T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8(+) T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNgamma, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8(+) T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8(+) T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8(+) T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.     

Mechanisms associated with defective TH1 cytokine production in HIV infection

Qualitative and quantitative changes in immune functions of different T-cell subsets associated with infection by human immunodeficiency virus type 1 (HIV-1) were analyzed by flow cytometric assessment of intracytoplasmic cytokines. The T(H)1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), were produced by both CD4 and CD8 T-cell subsets. When normal peripheral blood mononuclear cells (PBMC) were activated in culture, both cytokines were produced predominantly by CD4 (CD4) cell and only a minor fraction of normal CD8 cells produced these cytokines. In the cultures of PBMC from HIV-1-infected individuals (HIV+PBMC), more HIV+CD8 cells produced IL-2 and IFN-gamma. Production of IFN-gamma by HIV+CD4 cells was markedly reduced, while IL-2nd tumor necrosis factor-alpha (TNF-alpha) production by HIV+CD4 remained relatively intact until the disease progressed further. Normal CD4 cells which were isolated by using a cell sorter, FACSCalibur was still able to produce IL-2 and TNF-alpha. But for full production of IFN-gamma, normal CD4 required some accessory cells, the identity of which could not yet be established.     

Hemoglobin autooxidation/oxidation mechanisms and methemoglobin prevention or reduction processes in the bloodstream. Literature review and outline of autooxidation reaction

The human carcinoembryonic antigen (CEA), which is expressed in several cancer types is a potential target for antigen-specific immunotherapy. In this study, we show that dendritic cells (DC) pulsed with an HLA class I restricted CEA cytotoxic T lymphocyte (CTL) peptide epitope can stimulate T cells to kill CEA peptide loaded T2 target cells as well as CEA expressing tumor lines in the presence of interleukin-7 (IL-7) in an HLA-restricted manner. This has been demonstrated for carcinoma patients as well as healthy donors. The DC-CEA + IL-7 stimulated cultures contained predominantly CD3+CD8+CD56- cells indicative of MHC class I restricted CTL. In addition, DC-CEA + IL-7 stimulated cells showed higher levels of CD69 expression compared with cells stimulated with IL-7 alone, implying an activated phenotype. When the T-cell receptor (TCR) from CTL cultures stimulated with DC-CEA + IL-7 was analyzed, an oligoclonal pattern of expression was found for certain V beta subfamilies compared with the polyclonal patterns shown by IL-7 or phytohemagglutinin stimulated T cells from the same donors. This TCR restriction appeared to be maintained and enhanced after additional rounds of restimulation with DC-CEA + IL-7. The association between cytotoxicity and TCR restriction suggests that TCR analysis may be useful as an in vitro indicator to monitor alterations in the T-cell population in response to antigen-specific immunotherapies.     

Replication-competent chimeric lenti-oncovirus with expanded host cell tropism

Baboon bone marrow was grafted into human immunodeficiency virus type 1-infected patients in the course of recent trials for AIDS treatment. Since the baboon genome harbors multiple copies of an endogenous oncovirus, chimeric lenti-oncoviruses could emerge in the xenotransplant recipient. To analyze the potential replication competence of hybrid viruses between different genera of retroviruses, we replaced most of the env gene of simian immunodeficiency virus with the env gene of an amphotropic murine leukemia virus. The hybrid virus could be propagated in human T-cell lines, in peripheral blood mononuclear cells of rhesus macaques, and in CD4- B-cell lines. Because of the expanded cell tropism, the hybrid virus might have a selective advantage in comparison to parental viruses. Therefore, emerging chimeric viruses may be considered a serious risk of xenotransplantation. A note of caution is also suggested for the use of pseudotyped lentiviral vectors for human gene therapy.     

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.     

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.