Biomedical subjects

S C Knight

Publications and source records attributed to S C Knight.

At least 37 records · Page 2Linked to original sources

PubMed1999

Role of beta-chemokines in HIV-1 infection of dendritic cells maturing from CD34+ stem cells.

OBJECTIVES: To study the susceptibility to infection by different strains of HIV-1 viruses and the roles of chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and regulated-on-activation-T-expressed-and-secreted [RANTES]) in CD34+ stem cells maturing into dendritic cells (DC). DESIGN: It has been controversial whether CD34+ stem cells are susceptible to HIV-1 infection and whether high levels of beta-chemokines are beneficial for suppressing HIV-1 infection during DC maturation. These questions were addressed using different strains of HIV-1 and CD34+ stem cells taken from cord blood and cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) to generate mature DC. METHODS: CD34+ stem cells were exposed with M-tropic virus Ba-L or T-tropic viruses IIIB or Rut at day 1. Beta-chemokines were added to some cells before the virus and kept throughout the culture. Virus replication was measured throughout the maturation of these cells into CD1a+ DC and CD1a- CD14+ cells using enzyme-linked immunosorbent assay (ELISA) for p24, nested polymerase chain reaction (PCR) for env and intracellular p24 detection by flow cytometry. RESULTS: First, CD34+ stem cells acquired or were infected by live virus because maturing cells showed infection by both M- and T-tropic viruses. Second, the viruses replicated actively during the maturation of CD34+ stem cells toward CD1a+ DC and CD1a- CD14+ cells. Third, beta-chemokines suppressed infection by M-tropic virus Ba-L. And finally, beta-chemokines enhanced infection by T-tropic viruses IIIB and Rut. CONCLUSIONS: In addition to the initial anti-M-tropic virus effect by beta-chemokines, selective pressure on viruses may also result because of an increase in susceptibility to T-tropic virus. Caution should be taken when evaluating the effect of beta-chemokine receptor agonists in AIDS therapy.

Antigens, CD34↗

PubMed1999

Murine dendritic cells internalize Leishmania major promastigotes, produce IL-12 p40 and stimulate primary T cell proliferation in vitro.

Metacyclic Leishmania promastigotes (PM), transmitted by sand-fly bite, are likely to interact initially with cells of the dendritic cell (DC) lineage(s) in the epidermis or dermis. Epidermal Langerhans cells internalize L. major amastigotes (AM) and transport them to draining lymph nodes (Moll, H., Fuchs, H., Blank, C. and Röllinghoff, M., Eur. J. Immunol. 1993. 23: 1595) but little is known about the interaction of DC with PM. The present study demonstrates that DC are able to internalize PM and that the fate of the parasites within DC differs from that within macrophages (Mphi). DC took up small numbers of PM which did not differentiate into AM but appeared to be degraded; Mphi internalized large numbers of PM into parasitophorous vacuoles where they differentiated into AM. In response to direct stimulation with PM, DC from both C3H ("resistant" to L. major infection) and BALB/c ("susceptible") up-regulated production of IL-12 p40. In contrast, IL-12 production by Mphi was not detected. DC exposed to either metacyclic PM or PM culture supernatants were also able to stimulate proliferative responses in lymph node T cells from naive mice. These data indicate that DC have the capacity to promote protective Th1 immune responses in Leishmania infection and suggest that DC exposed to PM may be useful in immunotherapy and vaccination.

Animals↗

PubMed1999

Retrovirally induced switch from production of IL-12 to IL-4 in dendritic cells.

Dendritic cells (DC) in HIV-1 infection show a reduced capacity to stimulate primary T cell proliferation. Exposure of bone marrow-derived DC to Rauscher leukemia virus (RLV) provides a mouse model for studying retrovirally induced reduction in stimulatory capacity for T cells. Treatment with IL-12, a cytokine that promotes the development of Th1 cells, has been postulated as a treatment for AIDS and is effective at restoring cell-mediated immunity in mice infected with mouse AIDS virus or with RLV (see Knight, S. C. and Patterson, S., Annu. Rev. Immunol. 1994. 15: 593-615 for references). Here we studied the direct effect of RLV and of IL-12 on bone marrow-derived DC. Normal DC produced IL-12 and IL-10 and stimulated primary allogeneic T cell proliferation. Exposure of DC to RLV caused reduced production of IL-12, production of IL-4 was seen in DC for the first time and T cell stimulation was inhibited. Addition of IL-12 reinstated and enhanced IL-12 synthesis in RLV-treated DC, abrogated production of IL-10 and IL-4 and restored stimulatory activity. Manipulation of cytokine production in DC could be a stratagem that has evolved in the retrovirus to avoid stimulation of cellular responses.

Animals↗

PubMed1999

Human peripheral blood contains two distinct lineages of dendritic cells.

Human peripheral blood contains two populations of dendritic cells (DC) but their developmental relationship has not been established. Freshly isolated CD11c- DC possessed a lymphoid morphology, lacked myeloid markers but expressed lymphoid markers (CD4+ CD10+) whilst the CD11c+ DC were monocytoid in appearance and expressed myeloid markers. Although both populations were allostimulatory, only the CD11c+ DC were able to take up antigen. Irrespective of the culture conditions the CD11c- cells developed into CD11c- CD13- CD33- CD4+ CD1a- CD83+/- DC. In contrast, cultured CD11c+ cells developed the phenotype CD11c+ CD13+ CD33+/- CD4- CD1a+ CD83+ CD9+. Only the CD11c+ DC expressed macrophage colony-stimulating factor (M-CSF) receptor and gave rise to CD14+, esterase+, phagocytic macrophages when cultured in M-CSF. These data suggest that these two populations of DC represent distinct lineages of antigen-presenting DC.

Antigens, Surface↗

PubMed1999

Gene expression during differentiation of human dendritic cells from cord blood cd34 stem cells.

Human cord blood CD34(+)stem cells were allowed to differentiate in the presence of cytokines stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha) into functional CD1a+dendritic cells (DC). A maximum of 1.9 x 10(6) CD1a+ cells were separated from the cells generated from 1.2 x 10(6) CD34(+) stem cells from an individual donor. The percentage of CD1a+cells separated rose to a maximum of 27% at day 11 and fell to 8% at 21 days. Reverse transcription-polymerase chain reaction analysis showed that interleukin 2 receptor, interleukin 3 receptor, interleukin 6 receptor, interleukin 12 receptor (IL-12R) and signal transducer and activator of transcription (STAT) 3, STAT 4 mRNA was expressed in all CD1a+cell populations throughout and appears to be constitutive. Expression of IL-12RmRNA was unexpected in CD1a+DC normally considered to be of myeloid lineage. Expression of interleukin 12 (IL-12) p40 subunit mRNA was not detected. Intermittent expression of the IL-12p35 subunit and IL-4R mRNA suggested that gene expression is inducible, but not obviously correlated with progressive DC development. Expression of mRNA for a spectrum of cytokine receptors indicates that CD1a+DC have the potential to respond to a variety of maturational signals.

Antigens, CD1↗

PubMed1999

Interleukin-12 administration in retroviral infection of mice increases the potential to produce functional dendritic cells from bone marrow stem cells.

Rauscher leukaemia virus (RLV) infection in mice causes production of lymph node and skin dendritic cells (DC) that fail to stimulate a primary mixed leukocyte reaction (MLR). Treatment of mice with IL-12 around the time of infection results in DC with normal stimulatory function (N.J. Williams, J.J. Harvey, I. Duncan, R.F.G. Booth, S.C. Knight, Cell Immunol. 183 (1988) 121-130). Here we derived DC from mouse bone marrow by culture with granulocyte macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha) for 10-12 days; DC were generated from bone marrow cells taken from normal mice, from mice injected 15 days earlier with RLV or from those receiving RLV plus five daily doses of 100 ng of IL-12 starting 2 days before infection. Infection of the DC with RLV was assessed from nested PCR with doubling dilutions of DNA and the capacity of DC to stimulate a MLR was tested. DC derived from bone marrow of IL-12 treated animals showed at least twice the level of infection with RLV as those from non-treated animals although infection never exceeded 20% of the cells. DC derived from bone marrow of mice given RLV caused negligible stimulation of the MLR but those from mice additionally treated with IL-12 functioned normally. Thus, treatment of mice with IL-12 promoted the potential of stem cells taken 12 days after the last IL-12 injection to develop into functional DC despite increased infection with virus. Treatment of mice with IL-12 may have a long term effect on the potential growth of DC from stem cells which may contribute to the potency of this cytokine in promoting cell mediated immune responses.

Animals↗

PubMed1999

Subpopulations of peripheral blood dendritic cells show differential susceptibility to infection with a lymphotropic strain of HIV-1.

Blood dendritic cells (DC) express CD4 and are susceptible to HIV infection. By electron microscopy two morphologically distinct types of DC were identified in peripheral blood. Only one of these two types was susceptible to infection with a lymphotropic strain of HIV-1. By FACS two populations could be defined based on the expression of CD11c. The morphology of cultured FACS-purified CD11c negative DC was similar to that DC population shown to be susceptible to infection with the lymphotropic strain of HIV. Furthermore after several hours in culture CXCR4, the co-receptor for lymphotropic strains of HIV-1, was expressed at a significantly higher level on the CD11c negative DC than on the CD11c positive cells. This study suggests that there are subpopulations of DC that show differences in susceptibility to infection with some strains of HIV-1.

Dendritic Cells↗

PubMed1999

MHC class II molecules transferred between allogeneic dendritic cells stimulate primary mixed leukocyte reactions.

Presentation of antigen to T cells is generally restricted by MHC type but the mixed leukocyte reaction (MLR) was thought to involve direct stimulation by dendritic cells (DC) of allogeneic T cells. However, here we showed that DC bearing allogeneic MHC class II acted synergistically with responder-type DC. Removal of residual DC from 'purified' responder T cell populations was achieved using treatment with DC-specific antibody and complement. These DC-depleted cells showed a significantly reduced response to allogeneic DC which was restored by addition of DC syngeneic with responder T cells. The studies support the concept that a major component of the MLR is the secondary presentation of alloantigens acquired from stimulator DC by DC of responder type. To investigate the reasons why DC and not other cells stimulate an MLR, synergy between DC and other cell types was investigated. Synergy was found exclusively between DC; macrophages, B cells or L cells transfected with MHC class II molecules did not contribute. When allogeneic DC were mixed in culture, transfer of MHC molecules between DC was observed as assessed by flow cytometry. Freshly obtained cell-free supernatants from cultured DC contained MHC class II and stimulated primary allogeneic MLR. DC of responder type acquired allogeneic MHC molecules from the supernatants and stimulated proliferation in syngeneic T cells. The capacity of DC both to shed and to acquire MHC molecules may contribute to their potency in stimulating primary responses, and could explain why passenger DC within allografts provide a potent stimulus for graft rejection.

Animals↗

PubMed1998

Interleukin-12 restores dendritic cell function and cell-mediated immunity in retrovirus-infected mice.

The effects of IL-12 treatment on the defects in DC function and on the reduced cell-mediated immunity induced in mice infected with Rauscher leukemia virus (RLV) were studied. DC from RLV-infected mice failed to stimulate significant allogeneic T cell proliferation but T cells from RLV-infected mice showed normal responses to allogeneic DC. In RLV-infected mice treatment with 5 doses of 100 or 300 ng IL-12 around the time of infection resulted in DC that stimulated normal T cell proliferation. Treatment of mice with 300 ng IL-12 but not 100 ng reduced T cell responses. RLV-infected mice showed reduced delayed hypersensitivity to a contact sensitizer. Infected animals receiving the low dose of IL-12 which allowed normal DC and T cell function gave normal delayed hypersensitivity reactions; IL-12 thus resulted in both normal T cell stimulation by DC and cell-mediated immunity. A failure of T cell stimulation by DC is associated with immunosuppression in retrovirus infection and the enhanced capacity of DC to stimulate T cells after IL-12 treatment may be beneficial.

Animals↗

PubMed1998

Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation.

Primary proliferative T cell responses require stimulation with antigen-pulsed dendritic cells (Ag-DC). Here we show that for optimal stimulation, dendritic cells (DC) not exposed directly to antigen are also required. Ag-DC added to DC-depleted T cells caused negligible primary stimulation; adding back DC resulted in stimulation. These effects were seen using the contact sensitizer fluorescein isothiocyanate (FITC), FITC conjugated to ovalbumin (FITC-OVA) or influenza virus as antigens. DC co-cultured with Ag-DC (using FITC or FITC-OVA) acquired antigen indicating that antigen was transferred between DC. DC that acquired antigen secondarily were separated by cell sorting and stimulated primary T cell proliferation directly. DC were also pulsed with FITC, washed thoroughly and incubated overnight. Supernatants contained shed antigen since DC incubated in these supernatants acquired antigen as indicated by flow cytometry. DC acquiring the shed antigen also stimulated T cell proliferation although the stimulation was not as effective as that seen when cell contact between DC and antigen-bearing DC occurred. Thus, in primary stimulation, activation of T cells may occur when there is an antigen gradient between Ag-DC and DC and the mechanisms underlying these effects are now being sought. We propose that this unique interaction between antigen-presenting cells may be a paradigm for self/non-self discrimination.

Animals↗

PubMed1998

Increase in dendritic cell numbers, their function and the proportion uninfected during AZT therapy.

The effects of AZT treatment on the numbers, level of infection and function of peripheral blood dendritic cells (DC) were examined in patients with HIV infection. This was a cross-sectional study of patients before AZT treatment and up to 20 months after initiation of treatment. Numbers of DC separated by density gradients were below the normal range in patients before treatment, but increased between 3 and 12 months of treatment. The numbers of DC per provirus copy rose from around 100 cells to 5000 cells and this decrease in viral load in DC was significant between 3 and 20 months of treatment. The capacity of DC to stimulate allogeneic T cell proliferation was low before treatment and significantly higher between 6 and 12 months after the start of AZT. This study indicated that AZT treatment produced beneficial effects on DC by increasing their numbers, reducing the provirus load and increasing their function in stimulating T cells. These results support the thesis that the function of these potent antigen-presenting cells is important in development of immunological defects in AIDS, and that effects of AZT treatment on DC may provide a measure of its therapeutic effect.

Anti-HIV Agents↗

PubMed1998

IL-12 increases CD80 expression and the stimulatory capacity of bone marrow-derived dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells derived from CD34 bone marrow stem cells. They undergo a series of maturational steps that allow them to stimulate primary T cell responses. Several cytokines are known to contribute to this process. In this study murine DC maturing from bone marrow progenitors under the influence of granulocyte macrophage colony stimulating factor and tumour necrosis factor-alpha were found to produce IL-12 as measured by ELISA and by flow cytometry to detect intracellular cytokine. Administration of additional IL-12 from day 3 to 7 of culture altered the function and phenotype of DC; enhanced stimulation of T cell proliferation by DC in allogeneic mixed leukocyte reactions was associated with an increase in the surface expression of CD80 on DC. These effects were dose dependent, and were consistently seen with IL-12 at 25 ng/ml and were less marked with IL-12 at 50 ng/ml. These results show that IL-12 is both produced by DC and can increase their stimulatory capacity. The findings suggest that there may be an autocrine effect of IL-12 on DC maturation and function.

Age Factors↗

PubMed1998

Analysis of human immunodeficiency virus type 1 (HIV-1) variants and levels of infection in dendritic and T cells from symptomatic HIV-1-infected patients.

Dendritic cells (DC) are required to initiate primary cellular immune responses. Human immunodeficiency virus type 1 (HIV-1) infection of DC may be central to transmission and persistence of virus and in the pathogenesis of AIDS. In symptomatic HIV-1-infected patients the proportion of DC in the mononuclear cell population was reduced. Provirus load in the T cells was 3-100 times higher than in DC and there was no correlation between the levels of infection in the two cell types. Phylogenetic analysis of amino acids in the V3 loop and flanking regions indicated intermingling of sequences and thus provides the first evidence for transfer of virus between DC and T cells in vivo. In one of three patients analysed there were significant differences in amino acid residues in the V3 region. This may reflect reduced interactions between DC and T cells in infected individuals and for the existence of variants with a stronger tropism for DC, which could play a role in transmission by initiating infection in mucosal DC.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Protection against ascending infection of the genital tract by Chlamydia trachomatis is associated with recruitment of major histocompatibility complex class II antigen-presenting cells into uterine tissue.

A mouse model of ascending infection following intravaginal inoculation with a strain of Chlamydia trachomatis isolated from humans has been used to identify immune mechanisms associated with protection against genital infection. BALB/c and C3H mice differed in their susceptibilities to infection and inflammatory disease. In both mouse strains, ascension of the organism and recruitment of bone marrow-derived mononuclear leukocytes were evident in uterine tissue 1 week postinfection. By 3 weeks the organism had been cleared and inflammation had been resolved in the BALB/c mice, but both persisted in the C3H animals. In athymic nude BALB/c mice both the organism and inflammation persisted, indicating the influence of the hosts' immune response on the outcome of infection. Both BALB/c and C3H mice had a Th1 response in draining lymph nodes, with predominant production of gamma interferon and tumor necrosis factor alpha, low levels of interleukin-10, and no detectable levels of interleukin-4. However, the composition of the early uterine infiltrate differed in these two mouse strains. Cell surface labeling and analysis of light scatter properties by flow cytometry identified a population of large, CD45(+) major histocompatibility complex class II mononuclear cells, which were a prominent feature of the infiltrates in BALB/c mice but were present in significantly lower numbers in C3H mice. These cells expressed the costimulatory molecules CD86 and CD40 and stimulated allogeneic T cells, suggesting that these mononuclear cells are a population of antigen-presenting cells and that they may play a role in clearing antigen and protecting against inflammatory disease in BALB/c mice. An additional level of immunological control may thus exist in genital chlamydial infection.

Animals↗

PubMed1998

The in-vitro generation of dendritic cells from blast cells in acute leukaemia.

Dendritic cells (DC) are potent antigen-presenting cells responsible for the initiation of primary antigen-specific immune responses. In chronic myeloid leukaemia DC have been generated from Ph+ cells and these Ph+ DC are capable of stimulating cytolytic T-cell responses against the parent leukaemia cells. The prevalence of this phenomenon in acute leukaemia (AL) is unknown and we have therefore studied a variety of acute leukaemias to determine their potential for DC development. Peripheral blood mononuclear cells (PBMC) from 21 cases of AL were cultured in GM-CSF + TNF alpha. Of these cases, 15 were viable in culture and cells with typical DC morphology were observed in 12 of these 15 cases. DC growing in culture expressed either CDla and/or CD83 and were HLA-DR+ CD40+ CD80+ CD86+ typical of mature DC. In 9/12 cases the cultured cells possessed potent antigen-presenting capacity as measured in the allo-MLR. The malignant origin of the cultured DC was confirmed by FISH analysis in two cases (one 5q- and one Ph+ AL) and by persistent aberrant expression of CD19 in two cases of biphenotypic leukaemia. Functional DC may be derived from AL blasts in a significant number of patients and such DC may be capable of inducing leukaemia-specific immune responses with potential for clinically beneficial effects.

Acute Disease↗

PubMed1997

Primary proliferative responses to peptides of HIV Gag p24.

Primary proliferative responses can be initiated by adding dendritic cells pulsed with antigen to autologous T cells in 20-microliter hanging drop cultures. To identify primary T-cell epitopes of HIV gag, a series of 23 overlapping peptides, 15 amino acids long, spanning the p24 region were used. Significant proliferative responses were induced in cells from healthy HIV-negative donors by 11 of these peptides. One of two peptides that bound human leukocyte antigen (HLA)-A *0201 in a peptide-binding assay using the antigen-processing defective cell line T2 also induced a primary proliferative response. Primary T-cell proliferation was seen in response to some peptides of gag that have not previously been identified as T-cell epitopes in cells from infected individuals. These epitopes might be useful not only for vaccines in antigenically naive individuals but also might increase the breadth of immune responses in seropositive patients.

Amino Acid Sequence↗

PubMed1997

Mechanisms of loss of functional dendritic cells in HIV-1 infection.

Dendritic cells (DC) are lost from blood and skin during injection with HIV-1; those remaining show a reduced capacity to stimulate T cell proliferation [S. C. Knight, AIDS 10, 807-817]. Our recent studies investigate mechanisms underlying these effects. DC exposed to HIV-1 vitro can act as targets for cytotoxic T cells, although optimal killing was not obtained until DC were exposed to HIV-1 for 3 days. This cytotoxicity may provide a feedback mechanism by which DC that have presented antigens are removed. However, this effect could also contribute to the reduction in DC during persistent infection. We have also investigated the effect of exposure to HIV-1 on DC function. DC exposed to HIV-1 IIIB virus for 2 h stimulated primary proliferative and cytotoxic T cell responses in vitro; these effects may be similar to those occurring during the early activation of protective antiviral immunity in vivo. After exposure of DC to virus for 5 days, stimulation of allogeneic T cells was reduced. However, a different situation applied when using DC developed from CD34+ cord blood stem cells under the influence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha that were exposed at 24 h to the same virus. These DC showed low levels of infection similar to peripheral blood DC but in contrast stimulated normal allogeneic T cell proliferation. The capacity of DC exposed to HIV-1 to stimulate T cell proliferation or to show a blocked stimulatory capacity may thus depend not only on the length of the exposure to virus but also on the maturational state of the DC. Loss in DC numbers and function on exposure to HIV-1 may result in lower levels of stimulation of T cells, which in turn may be instrumental in reduction of T cell numbers.

Cell Differentiation↗

PubMed1997

Dendritic cells as targets for cytotoxic T lymphocytes.

Dendritic cells (DC) carry antigen into lymph nodes where they may cluster with CD4 and CD8+ lymphocytes and activate both subsets in the initiation of immune responses. Since DC do not leave the lymph nodes in the efferent lymph they may die within the lymph nodes. Another possibility is that they are targets for cytotoxic T cells (CTL) when expressing appropriate epitopes. This possibility was tested in vitro using human peripheral blood DC to stimulate the development of primary CTL in response to HIV-1 or one of its T-cell epitopes (e.g. env 111-126) and secondary CTL in response to type A influenza virus. Pooled CTL generated during six day cultures in 60 replicate 20 microliters hanging drops were tested in a conventional CTL assay. The HIV or HIV peptide stimulated CTL lysed HIV infected DC while the influenza-virus induced CTL killed DC targets infected with this virus. DC were not lysed significantly until they had been exposed to virus for 2-3 days and thus are not highly susceptible to lysis. However, killing of DC after 2-3 days infection with virus may be a feedback mechanism for removing antigen presenting cells after they have stimulated T cell responses. Removal of persistently infected DC by CD8+ CTL may also contribute to the reduction in DC numbers observed in blood and skin in HIV infection.

Antigens, Viral↗