Changes in dendritic cell subsets in the lymph nodes of rhesus macaques after application of glucocorticoids.

Corticosteroids are used therapeutically as potent immunosuppressive and antiinflammatory drugs for a broad spectrum of diseases. Although corticosteroids are known to inhibit the production of many cytokines in activated T cells, there is also evidence for increases in IL-4 and in some cases IFNgamma production. These conflicting results may be caused by contrary effects of corticosteroids on different cell types involved in immune regulation, for instance antigen presenting cells (APC) versus T cells. In the present study we simultaneously investigated the effect of local as well as systemic application of glucocorticoids (GCC) on the phenotype of APC in the skin as well as the lymph nodes in a model primate species, the rhesus macaque. Using a range of APC markers, including CD68, HAM56, HLA-DR, CD1a, p55, RFD-1, and costimulatory molecules CD40, CD80, and CD86 we document the close phenotypic resemblance of rhesus and human APC. We noted that topical GCC treatment specifically lead to a marked decrease in the number of CD1a expressing cells in the draining lymph nodes. However, the number of CD1a positive cells in peripheral lymph nodes was not affected by systemic GCC treatment. Importantly, by performing double staining of CD1a with RFD-1 we observed a shift in the expression pattern of these dendritic cell markers in the lymph nodes, with an increase in the number of RFD-1 single positive cells relative to CD1a single positive and CD1a/RFD-1 double positive cells. These findings suggest that GCC treatment results in the presence of phenotypically more mature APC.

The relative resistance of HIV type 1-infected chimpanzees to AIDS correlates with the maintenance of follicular architecture and the absence of infiltration by CD8+ cytotoxic T lymphocytes.

Lymphoid tissues are the focus of critical events in HIV pathogenesis. Persistent and high levels of virus production, extensive trapping of virus particles in germinal centers, and progressive degenerative changes in lymph node architecture are characteristics of progressive HIV-1 infection. Infiltrates of granzyme B- and TIA-expressing CD8+ "cytotoxic" T lymphocytes (CTLs) precede involution of germinal centers in humans who develop AIDS. Similar to humans, HIV-1 infection in chimpanzees is active and persistent. However, in contrast to humans, they remain relatively resistant to AIDS. Lymph node biopsies from chimpanzees infected with HIV-1 or a related chimpanzee lentivirus were studied for the level and pattern of virus expression, changes in lymphoid architecture, CD8+ T cell infiltrates and the presence or absence of CTL markers. In stark contrast to HIV-1-infected humans, lymph nodes from infected chimpanzees had little virus deposition in germinal centers and a paucity of virus-expressing cells. Although some of the lymph nodes examined from infected animals had moderate follicular hyperplasia with infiltrating CD8+ T cells, none had evidence of follicular fragmentation. Most importantly, in marked contrast to infected humans, CD8+ T cells infiltrating the germinal center were negative for the CTL marker granzyme B. This evidence suggests that the infiltration of CD8+ CTLs into the germinal centers of lymph nodes may be a key determinant in AIDS pathogenesis.

Cytotoxic T cells and neutralizing antibodies induced in rhesus monkeys by virus-like particle HIV vaccines in the absence of protection from SHIV infection.

HIV Pr55gag has in the absence of other viral components the capacity to self assemble in budding noninfectious virus-like particles (VLP). The immunological spectrum of the HIV-1IIIB gag-derived VLP was expanded either by stable anchoring of chimeric modified gp 120 on the surface of the VLP (type 1) or by replacing sequences of the Pr55gag precursor by the V3 loop and a linear portion of the CD4 binding domain (type 2). This noninfectious antigen delivery system was evaluated for immunogenicity and efficacy in rhesus macaques without adjuvants. Intramuscular immunization with both types of VLP induced high titers of gag-specific antibodies ranging from 1/8000 to 1/510,000 for type 1 VLP and from 1/4000 to 1/16,000 for type 2 VLP. Only animals immunized with type 1 VLP developed substantial endpoint titers of env-specific antibodies (1/2000-1/32,000) with a neutralizing capacity at serum dilutions of 1/32-1/128. Gag- and env-specific cytotoxic T lymphocyte (CTL) activity was induced by both types of VLP at similar levels. Four weeks after the last immunization animals were challenged intravenously with 20 MID50 of the cell free homologous envelope simian/HIV-1IIIB chimeric challenge stock Despite HIV-1-specific neutralizing and CTL responses, all vaccinated animals became infected.

Characteristics of primary infection of a European human immunodeficiency virus type 1 clade B isolate in chimpanzees.

The aim of the study was to select, from a panel of candidate European human immunodeficiency virus type 1 (HIV-1) clade B primary virus isolates, one isolate based on replication properties in chimpanzee peripheral blood mononuclear cells (PBMC). Secondly, to evaluate the in vivo kinetics of primary infection of the selected isolate at two different doses in two mature, outbred chimpanzees (Pan troglodytes). Four different low passage, human PBMC-cultured 'primary' HIV-1 isolates with European clade B consensus sequence were compared for their ability to replicate in vitro in chimpanzee versus human PBMC. The isolate which yielded the highest titre and most vigorous cytopathic effect in chimpanzee PBMC was evaluated for coreceptor usage and chosen for evaluation in vivo. Only the HIV-1Han2 isolate replicated in chimpanzee PBMC in vitro at detectable levels. This isolate was demonstrated to utilize CCR4, CCR5 and CXCR4 coreceptors and could be inhibited by beta-chemokines. Infection of chimpanzees was demonstrated by viral RNA and DNA PCR analysis, both in plasma as well as in PBMC and lymph node cells as early as 3 weeks after inoculation. Antibodies developed within 6 weeks and continued to increase to a maximum titre of approximately 12800, thereafter remaining in this range over the follow-up period of 2 years. Compared to cell line-adapted HIV-1 isolates there were slight but no dramatic differences in the kinetics of infection of chimpanzees with this particular primary isolate.

Comparison of the response to T-cell activation by integrated HIV-1 and HTLV-1 LTR-lacZ vectors.

Human Jurkat T-cell clones containing stably integrated HIV-1 LTR or HTLV-1 LTR/lacZ vectors were studied to compare the responses of integrated LTRs to T-cell activation. Responses were compared also with those obtained in parallel with Jurkat cells stably expressing lacZ under the control of the cellular enhancer element NF-AT of the IL-2 promoter. Activation induced via the cell surface TCR/CD3 complex or the CD28 receptor elicited responses from the LTR of HIV-1; however, HTLV-1 LTR-directed expression was not observed following triggering of these cell surface pathways. Mitogenic activation by elevation of intracellular calcium (Ca2+) levels along with protein kinase C (PKC) signals was required for optimal expression of the HIV-1 LTR and the NF-AT element; however, increased intracellular Ca2+ was inhibitory to PKC-mediated expression from the HTLV-1 LTR. Time course experiments revealed a sustained PKC-mediated response by the HTLV-1 LTR, which was detectable in the absence of Ca2+ as early as 6 hr following stimulation. In contrast to the HTLV-1 LTR, in time course experiments the HIV-1 LTR responded to stimulation by mitogenic activation of PKC in the absence and presence of Ca2+ and by antiCD3 with lacZ expression beginning as early as 3 hr poststimulation. These results suggest that the HTLV-1 LTR appears to be refractory to several cellular pathways which are upregulatory to the HIV-1 LTR.

Detection of human T-cell leukaemia virus 1 permissive cells using cell lines producing selectable recombinant virions.

A selectable retrovirus vector based on a full length HTLV-1 provirus clone, pCS-HTLV-1, was constructed by replacing the coding regions for tax, rex and the 3' region of env with the prokaryotic neomycin resistance gene under the control of the CMV promoter. This vector, pHTLV-1-CMVneo, was transfected into HTLV-1 infected human lymphocytes and fibroblasts. The production of recombinant virus by these cells was measured by the transfer of G418 resistance to target cells. Infection of target cells showed a preference for human lymphocytes in addition to two human fibroblast cell lines, Hos7 and RD4, and the African green monkey kidney cell line, Cos7. This system provides a method to study the cellular tropism of HTLV-1 and additionally provides a model to facilitate molecular studies of the natural events of HTLV-1 infection and integration.

Inhibition of apoptosis in T cells expressing human T cell leukemia virus type I Tax.

This study set out to determine whether T cell dysfunction associated with HTLV-I led to increased sensitivity of infected cells to apoptosis or, owing to their potential to develop ATL, if infected cells would become resistant to this process. To test this hypothesis we utilized the monoclonal antibody anti-APO-1, which has been demonstrated to induce apoptosis in human T cells. Human T cell lines expressing HTLV-I showed reduced susceptibility to anti-APO-1-induced apoptosis despite expression of high levels of cell surface APO-1. Cell-free supernatant of the Tax-expressing cell line C8166 and heat-inactivated supernatant of the HTLV-I-producing cell line MT2 transferred increased resistance to anti-APO-1 to susceptible Jurkat T cells. Susceptible T cells transfected with an HTLV-I Tax-expressing vector or treated with soluble Tax protein became less susceptible to anti-APO-1-induced cell death. Furthermore, primary human lymphocytes treated with soluble Tax were less susceptible to apoptosis induced by anti-APO-1. The protective effect of Tax in T cell lines and primary human lymphocytes was reversed by the addition of anti-Tax antibodies. Anti-APO-1-induced apoptosis was also found to be inhibited in Jurkat cells by the induction of protein kinase C (PKC) with 12-O-tetradecanoylphorbol-13-acetate (TPA). Resistance to apoptosis conferred by HTLV-I Tax and an active PKC pathway may be factors contributing to the survival of dysregulated HTLV-I-infected T cells prone to the development of adult T cell leukemia.

Calcium-mediated inhibition of phorbol ester and Tax trans-activation of the human T cell leukaemia virus type 1.

Human Jurkat T cells containing a stably integrated human T cell leukaemia virus type 1 (HTLV-1) long terminal repeat (LTR) reporter gene construct were used to study the role of calcium-dependent cellular activation pathways in LTR trans-activation. Treatment of these cells with the calcium ionophore ionomycin resulted in a reduced basal response of the LTR and reduced responses to 12-O-tetradecanoylphorbol-13-acetate-and Tax-mediated trans-activation. This effect was also observed for virus production in the HTLV-1-producing T cell line MT-2. Experiments designed to determine the events underlying this inhibition, using inhibitors of calcium-related events, revealed that the ionomycin-induced repression of the LTR was alleviated in all cases by cyclosporin A. This compound was also effective in preventing the ionomycin-induced reduction in virus production in MT-2 cells. These results suggest a role for calcium-related events in the down-regulation of HTLV-1 expression.

Cytochemical analysis of human T cell leukaemia virus I LTR-regulated beta-galactosidase gene expression using a novel integrated cell system.

To develop a reporter system to study the response of an integrated retroviral LTR and cellular and viral events which influence transcription, the 5' LTR of HTLV-1 was coupled to the Escherichia coli beta-galactosidase gene (lacZ). This construct was assembled within a vector containing the neomycin resistance gene controlled by the SV40 promoter, and introduced into HeLa cells. Expression from the LTR in one clone was upregulated by positive regulators of HTLV-1 expression, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and the HTLV-1 transregulatory protein (tax), as has been previously reported using transient transfection assays. This method proved to be a rapid and reproducible assay for the measurement of integrated viral LTR activation in a single cell system.

Lymphoproliferative disorders developing after transplantation and their relation to simian T-cell leukemia virus infection.

In this report the role of the HTLV-1-like simian T-cell leukemia virus (STLV) during the development of posttransplantation lymphoproliferative disorders (PTLPD) is described. To prevent rejection of an allogeneic transplant in 12 rhesus monkeys cyclosporin A (CyA), prednisone, and/or lymphocyte-specific monoclonal antibodies were used for immunosuppression. Seven monkeys died during the experiment between 22 and 179 days postoperatively. At autopsy in 4 monkeys PTLPD were found. In each case, STLV provirus was acquired during the experiment, either from the blood transfusions or allograft donors. Seroconversion of anti-STLV titers occurred in 3 monkeys. However, Southern blot analysis showed the presence of STLV provirus at the DNA level in all PTLP tissues. PTLPD morphology and phenotype varied significantly. In conclusion, for the first time the oncogenic potential of STLV is identified in a rhesus monkey transplantation model. Moreover, the importance of screening blood and organ donors for HTLV-1 must be emphasized.