Simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocytes in gastrointestinal tissues of chronically SIV-infected rhesus monkeys.

Although systemic virus-specific cytotoxic T lymphocyte (CTL) responses are of critical importance in controlling virus replication in individuals infected with human immunodeficiency virus 1 (HIV-1), little is known about this immune response in the gastrointestinal (GI) tract. This study investigated the GI tract CTL response in a nonhuman primate model for HIV-1 infection, simian immunodeficiency virus (SIV)-infected rhesus monkeys. Lymphocytes from duodenal pinch biopsy specimens were obtained from 9 chronically SIVmac-infected rhesus monkeys and GI tract lymphocytes were harvested from the jejunum and ileum of 4 euthanized SIVmac-infected rhesus monkeys. Lymphocytes were also assessed in GI mucosal tissues by in situ staining in histologic specimens. SIVmac Gag-specific CTLs were assessed in the monkeys using the tetramer technology. These GI mucosal tissues of chronically SIVmac-infected rhesus monkeys contained levels of CTLs comparable to those found in the peripheral blood and lymph nodes. The present studies suggest that the CD8(+) CTL response in GI mucosal sites is comparable to that seen systemically in SIVmac-infected rhesus monkeys.     

Simian immunodeficiency virus-specific T-cell-mediated proliferative response of infected rhesus macaques

While cell-mediated immunity is known to play an important role in controlling viral infections, its role in human and experimental animal models of human AIDS has not been established. To address this issue, four juvenile rhesus macaques were infected with simian immunodeficiency virus SIVMAC. Freshly isolated peripheral blood mononuclear cells from these SIVMAC-infected macaques and four uninfected control macaques were assessed for T-cell proliferative activity to SIV, monthly, for 10 consecutive months. T cells from SIV-infected monkeys failed to proliferate in response to SIV added directly to the culture. However, when SIV was processed by autologous antigen-presenting cells prior to culture with purified T cells, proliferative responses were uniformly demonstrated in SIV-infected monkeys, but not in uninfected controls. Proliferation in response to heat-inactivated SIV was mediated by CD4+ T cells and was shown to be MHC class II-restricted. However, the proliferative response to infectious SIV was mediated by both CD4+ and CD8+ T cells and was MHC class-restricted. As disease progressed, a decline in the T-cell proliferative response was observed.     

Characterization of simian immunodeficiency virus-specific T-cell-mediated cytotoxic response of infected rhesus macaques

Four juvenile rhesus macaques were infected with simian immunodeficiency virus (SIV)MAC-Freshly isolated peripheral blood mononuclear cells (PBMC) from these SIVMAC-infected and from uninfected control macaques were assessed for cytotoxic T-lymphocyte (CTL) activity monthly for 7 consecutive months, beginning 2 months after infection. Target cells consisted of major histocompatibility complex (MHC) haploidentical parental PBMC which were stimulated with mitogen and then pulsed with heat-killed SIVMAC. CTL activity was demonstrated on all four infected animals. The effector cells are T cells which mediate cytotoxicity against SIVMAC-pulsed target cells in an MHC-restricted manner. Furthermore, the cytotoxicity is virus specific and predominantly, if not exclusively, mediated by CD8+ T cells; it is also MHC class-I restricted. Incubation of target cells with leupeptin prior to the cytotoxic assay inhibited target cell generation, suggesting that viral antigens are processed via an endocytic pathway.     

Inverse correlation between memory Gag-specific cytotoxic T lymphocytes and viral replication in human immunodeficiency virus-infected children

A previous study showed that, during the first year of life, the presence of cytotoxic T lymphocytes (CTLs) in human immunodeficiency virus (HIV)-infected children is associated with a lack of rapid progression to acquired immunodeficiency syndrome. The goal of the study was to address the role of CTLs in children who survived after age 5 years. Memory HIV-specific CTLs directed against Env, Gag, Nef, and Pol proteins were measured in a group of 47 highly active antiretroviral therapy-naive HIV-infected children. Both Gag- and Pol-specific CTLs were positively correlated with CD4(+) T cell counts. Gag-, Nef-, and Pol-specific CTLs were inversely correlated with virus load. The inverse correlation between virus load and Gag-specific CTLs was independent of CD4(+) T cell counts. In conclusion, this study showed the beneficial role of HIV-specific CTLs in children who survived after age 5 years.     

Longitudinal dynamics of Epstein-Barr virus-specific cytotoxic T lymphocytes during posttransplant lymphoproliferative disorder

Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD) is a serious complication after allogeneic bone marrow transplantation (BMT). Dynamics of EBV-specific cytotoxic T lymphocytes (CTL), which are important in controlling EBV during the LPD, have not been fully elucidated. A patient with Wiskot-Aldrich's syndrome was diagnosed as suffering from LPD on day 47 after BMT. Fluorescence-activated cell sorter (FACS) analysis for interferon-gamma production revealed that >70% of the patient's CD8(+) T cells were EBV specific. The patient's lymphocytes were directly cytotoxic to donor-derived EBV-positive lymphoblastoid cells, which was blocked by an anti-class I antibody. EBV-specific CD8(+) T cell counts declined in parallel with EBV genome load, and full recovery of LPD was obtained with relaxation of immunosuppressive drugs. The results illustrate longitudinal dynamics of EBV-specific CTL during the posttransplant LPD; they also illustrate the advantages of using FACS analysis for EBV-specific CTL to make decisions about treatment.     

Early activation and proliferation of T cells in simian immunodeficiency virus-infected rhesus monkeys

To longitudinally determine T cell activation and turnover in early simian immunodeficiency virus (SIV) infection of macaques, immunological and virological parameters were monitored in 10 SIV-infected animals starting before infection until 40 weeks postinfection (wpi). Lymphocyte subsets in blood and lymph nodes (LNs) were characterized by three-color flow cytometry for expression of markers of activation, proliferation, and differentiation. As early as 1 wpi, CD69 expression was upregulated both on CD4+ and CD8+ T cells, indicative of an early activation of these cells. Whereas this activation led to increased proliferation, determined by expression of Ki-67, and absolute numbers of CD8+ T cells, CD4+ T cells showed a decreased expression of Ki-67 and reduced counts in blood at 2 wpi. Later, the percentage of Ki-67-expressing CD4+ T cells in blood and LNs increased again above preinfection levels in most animals but remained low in two monkeys progressing to AIDS. These findings suggest that T cells are activated after SIV infection, leading to increased T cell proliferation already in the early asymptomatic phase. In addition, we found a correlation between the capacity to regenerate CD4+ T cells by peripheral proliferation and the disease course. Moreover, our data indicate that the increased peripheral T cell proliferation during immunodeficiency virus infection is probably not caused by the effort of the immune system to maintain T cell homeostasis but may be a reflection of the ongoing immune response against the virus.     

Generation of hepatitis C virus-specific cytotoxic T lymphocytes from healthy individuals with peptide-pulsed dendritic cells

BACKGROUND AND AIMS: In hepatitis C virus (HCV) infection, cytotoxic T lymphocytes (CTL) are involved in liver inflammation and contribute to the reduction of viral load. Antibodies for HCV-CTL precursor frequencies (CTLpf) are relatively low in chronic hepatitis C, and this may be related to the poor CTL response in vivo. The aim of this study was to assess the efficacy of dendritic cells (DC) as antigen-presenting cells in CTL generation from low CTLpf. METHODS: To confirm the rationale of using DC to prime naive T cells, five HCV-uninfected individuals were enrolled in the study. We obtained DC by maturation from peripheral progenitors under stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and IL-1alpha. Autologous T cells were cultured with DC or concanavalin-A-induced blasts loaded with four HCV-derived peptides bearing human leukocyte antigen (HLA)-A*0201 or -A24 motifs for 28 days under IL-7 and IL-2 stimulation. The lytic activity against peptide-pulsed targets was assessed by using a [51Cr]-releasing assay. RESULTS: The DC strongly expressed HLA class I, II, B7-1 and B7-2, but not phenotypic markers of T-, B-, natural killer (NK)-cells or monocytes. The CD8-positive, HLA-class I-restricted and HCV peptide-specific CTL were generated with DC from HLA-A antigen-matched subjects, whereas no CTL activity was detected with concavalin (Con-A) blasts. We were thus able to generate HCV specific CTL from naive precursors with peptide-pulsed DC. CONCLUSIONS: This DC-based system can be used to generate CTL of desired antigen specificity, even from a source with low CTLpf.     

Simian immunodeficiency virus (SIVsm) infection of cynomolgus monkeys: effects on follicular dendritic cells in lymphoid tissue.

We studied follicles in sections of lymph nodes and spleen from cynomolgus monkeys (Macaca fascicularis) after infection with simian immunodeficiency virus (SIVsm), by (immuno)histology and (immunogold) electron microscopy. Also isolated follicular dendritic cells (FDC) were investigated. Histology showed ranged from follicular hyperplasia to follicle fragmentation. FDC showed desmin and vimentin, characteristic of mesenchymal cells. Except for two animals who got experimental chemotherapy in the first postinfection period, the cells expressed SIV gag p28 protein. Electron microscopy showed SIVsm-like particles in the germinal centers. A number of cell types in the germinal center, including FDC, showed tubuloreticular structures, indicative of alpha-interferon synthesis during an antiviral response. In immunogold electron microscopy, SIV p28 label was observed on the surface of FDC, on SIVsm-like particles, and in the cytoplasm of macrophages. A relatively high density of CD8-positive cells (T cytotoxic-suppressor phenotype) was observed around and in germinal centers, especially areas depleted of FDC. Cells immunoreactive for serine esterase granzyme-B, a protein occurring in granules of cytotoxic cells, occurred around germinal centers, but not in germinal centers at areas where FDC and SIV p28 label localized. This argues against a role of cytotoxic T cells in mediating follicle destruction.     

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.     

Expansion after epitope peptide exposure in vitro predicts cytotoxic T lymphocyte epitope dominance hierarchy in lymphocytes of vaccinated mamu-a*01+ rhesus monkeys

Because of the importance of developing HIV vaccine strategies that generate cytotoxic T lymphocyte (CTL) responses with a maximal breadth of epitope recognition, we have explored a variety of novel strategies designed to overcome the usual propensity of CTLs to focus recognition on a limited number of dominant epitopes. In studies of rhesus monkeys expressing the Mamu-A*01 MHC class I allele, we show that variously configured multiepitope plasmid DNA vaccine constructs elicit CTL populations that do not evidence skewing of recognition to dominant epitopes. Nevertheless, repeated boosting of these vaccinated monkeys with different live recombinant vaccine vectors uncovers and amplifies the usual CTL epitope dominance hierarchy. Importantly, in vitro peptide stimulation of peripheral blood mononuclear cells from monkeys that have received only a multiepitope plasmid DNA priming immunization uncovers this dominance hierarchy. Therefore, the dominance hierarchy of the vaccine-elicited epitope-specific CTL populations is inherent in the T lymphocytes of the monkeys after initial exposure to epitope peptides, and the ultimate breadth of epitope recognition cannot be modified thereafter. This finding underscores the enormous challenge associated with increasing the breadth of CTL recognition through vaccination.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs)

We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-gamma enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity.     

Detection of viral RNA in CD4(-)CD8(-) and CD4(-)CD8(+) lymphocytes in vivo in rhesus monkeys infected with simian immunodeficiency virus of macaques

A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.     

Priming of hepatitis C virus-specific cytotoxic T lymphocytes in mice following portal vein injection of a liver-specific plasmid DNA

The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8(+) cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with "naked" DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.     

CD8+ T lymphocytes of African green monkeys secrete an immunodeficiency virus-suppressing lymphokine.

Following natural and experimental infection by simian immunodeficiency virus SIVagm of African green monkeys (AGMs), the natural host, there is no evidence for the development of an immunodeficiency. Within the framework of our studies on human immunodeficiency virus (HIV)/SIV pathogenesis, we investigated the influence of CD8 T lymphocytes on SIVagm replication in AGM CD4 T lymphocytes in vitro. The following observations were made: (i) Peripheral blood mononuclear cells from both seronegative and seropositive AGMs contained only a low proportion (i.e., 10%) of CD4+ lymphocytes, whereas a high proportion (80%) of CD8+ cells was detected. Even after persistent SIVagm infection, CD4 T lymphocytes do not decrease in number. (ii) The target of in vitro infection of peripheral blood cells is the CD4+ mononuclear cell (T lymphocytes, monocytes) and SIVagm infects by binding to the CD4 molecule. (iii) In both naturally and experimentally SIVagm-infected AGMs the CD4+ T cells and monocytes, but not the CD8+ T cells, harbor DNA provirus. (iv) Virus reisolation and virus replication of SIVagm in CD4 T lymphocytes from seropositive AGMs is suppressed in the presence of autologous CD8 T lymphocytes or a soluble factor produced by these cells. Taken together, one possible reason for the apathogenicity of the SIVagm infection in AGMs may be the suppression of virus replication by a soluble, yet unidentified factor secreted by CD8 lymphocytes quantitatively dominating among peripheral blood cell populations. We have tentatively termed this factor "immunodeficiency virus-suppressing lymphokine." In addition, we show that immunodeficiency virus-suppressing lymphokine from AGMs is able to suppress HIV-1 replication in human CD4+ T cells.     

Induction of cytotoxic T lymphocytes by dendritic cells pulsed with murine leukemic cell RNA

Peptide-pulsed dendritic cells can stimulate T cells showing specific cytotoxicity in chronic myelogenous leukemia. We tried to induce a specific cytotoxic T-cell response stimulated by RNA-pulsed dendritic cells in acute myelogenous leukemia. The total RNA of WEHI-3BD+, a myelomonocytic leukemia cell line derived from BALB/c mice, was transfected into dendritic cells induced from bone marrow nucleated cells of BALB/c mice with granulocyte macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) using liposome. RNA-pulsed dendritic cells were injected into the peritoneal cavity of BALB/c mice, and splenic T cells were isolated for antigen-stimulated proliferation and leukemia-specific cytotoxicity assay. Cultured bone marrow nucleated cells expressed dendritic cell markers including MHC class II antigen, CD80, CD86, and CD11c. T cells stimulated by RNA-pulsed dendritic cells showed enhanced proliferation than those stimulated by unpulsed dendritic cells (P = 0.05) and showed dose-dependent specific cytotoxicity against WEHI-3BD+ cells. We concluded total RNA-pulsed dendritic cells could induce a specific T-cell cytotoxicity in acute myelogenous leukemia.     

Cytokine production and cytolytic mechanism of CD4(+) cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus-specific T-cell cultures

Ex vivo expanded Epstein-Barr virus (EBV)-specific T cells have been successfully applied clinically for adoptive immunotherapy. However, the role of CD4(+) T cells in the therapeutic T-cell culture has not been established for the reconstitution of EBV-specific immunity. We isolated and characterized CD4(+) T-cell lines from the ex vivo T-cell cultures. Monoclonal line PD-F4 and oligoclonal lines ND-R4 and TD-B4 were CD3(+)CD4(+)CD8(-). Cytolytic tests with targets of mismatched major histocompatibility complex (MHC) and anti-MHC antibodies confirmed that the cytotoxicity of these CD4(+) cells was restricted by MHC class II. Single cells of ND-R4 expressed interferon-gamma (IFN-gamma, or interleukin 4 (IL-4), but rarely coexpressed these 2 cytokines. In contrast, PD-F4 coexpressed IFN-gamma, IL-2, and IL-4. Kinetic studies with PD-F4 showed that expression of the 3 cytokines plateaued 5 hours upon stimulation and was then drastically reduced, with a pattern consistent with independent modulation and differential off-cycle signal requirements. The cytotoxicity of these CD4(+) cells was largely resistant to brefeldin A, an inhibitor for cytolytic pathways by Fas-ligand family molecules. Although sensitive to concanamycin A and ethyleneglycotetraacetic acid, which inhibit cytotoxicity by granule exocytosis, the CD4(+) cytotoxic T lymphocytes (CTLs) did not express perforin, suggesting a cytotoxic mechanism independent of perforin although involving exocytosis. Flow cytometric analysis showed that the CD4(+) CTLs expressed granulysin, a recently identified cytolytic molecule associated with exocytotic cytolytic granules. These data suggested that CD4(+) T cells in the therapeutic B-lymphoblastoid cell lines-primed T-cell culture are diverse in producing T(H)1 and T(H)2 cytokines, and may exert specific cytotoxicity via exocytosis of granulysin.