David S. Ritchie

CD8+ T Cell-Dependent Elimination of Dendritic Cells In Vivo Limits the Induction of Antitumor Immunity

The fate of dendritic cells (DC) after they have initiated a T cell immune response is still undefined. We have monitored the migration of DC labeled with a fluorescent tracer and injected s.c. into naive mice or into mice with an ongoing immune response. DC not loaded with Ag were detected in the draining lymph node in excess of 7 days after injection with maximum numbers detectable ∼40 h after transfer. In contrast, DC that had been loaded with an MHC class I-binding peptide disappeared from the lymph node with kinetics that parallel the known kinetics of activation of CD8+ T cells to effector function. In the presence of high numbers of specific CTL precursors, as in TCR transgenic mice, DC numbers were significantly decreased by 72 h after injection. The rate of DC disappearance was extremely rapid and efficient in recently immunized mice and was slower in “memory” mice in which memory CD8+ cells needed to reacquire effector function before mediating DC elimination. We also show that CTL-mediated clearance of Ag-loaded DC has a notable effect on immune responses in vivo. Ag-specific CD8+ T cells failed to divide in response to Ag presented on a DC if the DC were targets of a pre-existing CTL response. The induction of antitumor immunity by tumor Ag-loaded DC was also impaired. Therefore, CTL-mediated clearance of Ag-loaded DC may serve as a negative feedback mechanism to limit the activity of DC within the lymph node.

Late Ebola virus relapse causing meningoencephalitis: a case report

Summary Background There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13·2). Methods A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach. Findings On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23·7) than plasma (31·3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus. Interpretation Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern. Funding Royal Free London NHS Foundation Trust.

Dendritic cell elimination as an assay of cytotoxic T lymphocyte activity in vivo.

We show in this paper that the survival of antigen-loaded dendritic cells in vivo may be used as a sensitive readout of CTL activity. We have previously shown that dendritic cells labeled with the fluorescent dye CFSE and injected sub-cutaneously into mice migrate spontaneously to the draining lymph node where they persist for several days. In the presence of effector CTL responses, dendritic cells loaded with specific antigen rapidly disappear from the draining lymph node. In this paper we extend the above observations and set up a simple and sensitive method to reveal CTL activity in individual mice in vivo. Dendritic cells were labeled with two different fluorochromes, loaded with antigen or left untreated, and mixed together before injection into mice. We show that only the dendritic cells loaded with specific antigen were cleared from the draining lymph node, while dendritic cells not loaded with antigen remained unaffected. Cytotoxic responses generated by immunization with peptide-loaded dendritic cells, or by infection with influenza virus, could be revealed using this method. Comparison of the differential survival of dendritic cells populations mixed together also allowed us to accurately evaluate the disappearance of dendritic cells, irrespective of variability in the injection site and other parameters. Given the ability of dendritic cells to efficiently take up and present complex antigens, nucleic acids and apoptotic bodies, this method may also allow the evaluation of cytotoxic activity against antigens that are not characterized in terms of peptide epitopes.

B-Lymphocytes activated by CD40 ligand induce an antigen-specific anti-tumour immune response by direct and indirect activation of CD8(+) T-cells.

In this report, we describe the ability of CD40‐ligand (CD40L)‐activated, antigen‐loaded B‐cells to initiate antigen‐specific anti‐tumour immune responses in vivo. Mice immunized by means of intravenous administration of CD40L‐activated B‐cells loaded with an MHC class‐I‐binding peptide, and challenged with a tumour cell line expressing the same class‐I epitope, showed a marked delay in tumour growth, compared to non‐immunized controls or to mice receiving either freshly isolated B‐cells or B‐cells activated with lipopolysaccharide or interleukin‐4.

Dendritic Cells Treated with Lipopolysaccharide Up-Regulate Serine Protease Inhibitor 6 and Remain Sensitive to Killing by Cytotoxic T Lymphocytes In Vivo

Ag presentation by dendritic cells (DC) in vivo is essential to the initiation of primary and secondary T cell responses. We have reported that DC presenting Ag in the context of MHC I molecules also become targets of specific CTL and are rapidly killed in mice. However, activated DC up-regulate expression of serine protease inhibitor (SPI)-6, a specific blocker of the cytotoxic granule protein granzyme B, which modulates their susceptibility to CTL-mediated killing in vitro. We wanted to determine whether susceptibility to CTL-mediated killing in vivo is also modulated by DC activation. As was previously reported by others, DC treated with different doses of LPS expressed higher levels of SPI-6 mRNA than did untreated DC. The increased expression of SPI-6 was functionally relevant, as LPS-treated DC became less susceptible to CTL-mediated killing in vitro. However, when these LPS-treated DC were injected in vivo, they remained sensitive to CTL-mediated killing regardless of whether the CTL activity was elicited in host mice via active immunization or was passively transferred via injection of in vitro-activated CTL. LPS-treated DC were also sensitive to killing in lymph node during the reactivation of memory CTL. We conclude that increased SPI-6 expression is not sufficient to confer DC with resistance to direct killing in vivo. However, SPI-6 expression may provide DC with a survival advantage in some conditions, such as those modeled by in vitro cytotoxicity assays.

Tumour antigens on tap

See page 273 In this issue of The Lancet, Anthony Swerdlow and colleagues report findings on cancer risk in a cohort of 1848 patients who were deficient in growth hormone and treated with human-pituitary growth hormone at young ages between 1959 and 1985. These individuals were followed up for cancer incidence to December, 1995, and for mortality to December, 2000. Compared with cancer rates in the general population, rates in treated patients showed significantly increased risks of mortality from cancer overall, particularly from colorectal cancer and Hodgkin’s disease. The investigators are rightly cautious in their interpretations of the findings. Several points, mostly

Autologous dendritic cells pulsed with eluted peptide as immunotherapy for advanced B-cell malignancies

We have studied the feasibility, safety and efficacy of vaccination with autologous dendritic cells pulsed with eluted peptide in patients with advanced low-grade B-cell malignancies. This study demonstrates that autologous dendritic cell vaccines can be successfully produced from patients with advanced disease and be delivered without significant toxicity. Furthermore, we have demonstrated immunological and clinical responses in two of ten patients treated. These results provide further evidence for the use of immunotherapy in the management of B-cell malignancies, but also suggest that sustained responses may only be possible in patients with low bulk disease early in the disease course.