Marie Larsson

Primary Tumor Tissue Lysates Are Enriched in Heat Shock Proteins and Induce the Maturation of Human Dendritic Cells

Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.

A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy

Dendritic cells (DCs) can induce tumor- or pathogen-specific T cell responses in humans. We comprehensively compared the clinically available DC maturation stimuli for their ability to promote uniformly mature DCs that elicit higher levels of T cell responses. We compared the standard maturation stimulus, autologous monocyte-conditioned medium (MCM), with a synthetic double stranded RNA (poly I:C), soluble CD40 ligand trimer, and a defined cocktail of cytokines (TNF-alpha, IL-1 beta, IL-6) and PGE(2) to promote mature phenotype and function in human monocyte-derived DCs. The cocktail was the most efficient despite the lack of induction of IL-12p70. While these results support the use of the MCM-mimic cocktail in clinical DC immunotherapy trials, the roles of its individual constituents remain to be completely defined.

Generation of high quantities of viral and tumor-specific human CD4+ and CD8+ T-cell clones using peptide pulsed mature dendritic cells.

CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-gamma intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy.

Strong Human Immunodeficiency Virus (HIV)-Specific CD4+ T Cell Responses in a Cohort of Chronically Infected Patients Are Associated with Interruptions in Anti-HIV Chemotherapy

Virus-specific CD4+ T-helper cell function is important in controlling human immunodeficiency virus (HIV) infection but is impaired in patients with progressive HIV disease. It has been reported that after highly active antiretroviral therapy (HAART), HIV-specific lymphoproliferative responses remain absent, whereas responses to non-HIV microbial antigens are restored. However, in analyzing immune responses in a cohort of chronically infected adults on HAART, we observed strong HIV-specific CD4+ T cell responses of Th-1 phenotype in 11 of 22 patients. The magnitude and frequency of HIV-specific lymphoproliferative responses was strongly associated with previous interruptions in HAART (P=.001). In contrast, the magnitude of CD8+ T cell responses to HIV Gag, Pol, Env, and Nef was similar in patients who had and those who had not interrupted HAART. We conclude that (1) a significant proportion of chronically HIV-infected patients on HAART can generate strong HIV-specific CD4+ and CD8+ T cell immunity and (2) transient interruptions in antiviral treatment may prime or boost HIV-specific CD4+ T-helper responses.

Interactions between dead cells and dendritic cells in the induction of antiviral CTL responses.

Dendritic cells - professional antigen-presenting cells - are key players for activating adaptive immune responses against viruses. Apoptosis or lytic cell death often accompanies viral infection. Dendritic cells can acquire infected dead or dying cells as exogenous sources of antigens for presentation on MHC class I and II molecules to initiate T cell responses. This pathway of activating T cells may be critical for the development of effective antiviral immunity in vivo.

Changes in Frequency of HIV-1—Specific Cytotoxic T Cell Precursors and Circulating Effectors after Combination Antiretroviral Therapy in Children

Combination antiretroviral therapy has had a major role in reducing human immunodeficiency virus type 1 (HIV-1) plasma viral loads in HIV-1-infected adults but a variable effect in infants, in whom complete viral suppression appears to be less readily achieved. In adults, after the reduction in plasma viremia, there is a decrease in the numbers of circulating cytotoxic T cell (CTL) effectors and precursors in the majority of patients. This longitudinal study assessed the effect of combination drug therapy on the frequency of HIV-1-specific CTL responses in 8 HIV-1-infected children. Following treatment, the frequency of HIV-1-specific CTL responses initially increased, especially in children with incomplete viral suppression but with increasing CD4+ cell counts. In children with complete viral suppression, the frequency of HIV-1-specific CTL responses decreased, suggesting that viral replication is required to maintain CTL responses in the systemic circulation.

Presentation of Epstein‐Barr virus latency antigens to CD8+, interferon‐γ‐secreting, T lymphocytes

Epstein‐Barr virus (EBV) infects more than 95 % of the human population and causes an asymptomatic life‐long infection in the majority of EBV carriers. Cell‐mediated immunity provides resistance to EBV, as demonstrated by the occurrence of EBV‐induced post‐transplant lymphoproliferative disease in immunosuppressed patients. Here we looked for IFN‐γ‐producing T lymphocytes in the blood of healthy donors with a rapid enzyme‐linked immunospot (ELISPOT) assay, comparing as antigen presenting cells monocytes and dendritic cells (DC) infected with recombinant vaccinia virus (rVV). We found a strong CD8+ ELISPOT response to one or more of the EBNA 3A, 3B and 3C antigens in the PBMC from 14 / 18 donors. The sensitivity of the overnight ELISPOT assay was increased using DC as antigen‐presenting cells, including 3 / 3 individuals who lacked ELISPOT in PBMC. In addition, DC could markedly expand EBV‐specific spots after a 7‐day culture. In a smaller number of donors, we documented recognition of the subdominant LMP 1, LMP 2 and EBNA 1 antigens that are expressed in a variety of EBV‐associated malignancies. Therefore our data provide more evidence for the efficacy of DC in eliciting rapid responses to EBV latency antigens in circulating CD8+ T cells.

Amplification of low-frequency antiviral CD8 T cell responses using autologous dendritic cells

Objective To utilize the potent antigen-presenting capacity of mature dendritic cells (MDC) in order to develop a rapid, sensitive method for quantifying antigen-specific CD8 T cells present at low frequency in peripheral blood. Design Peripheral blood mononuclear cells (PBMC) were obtained from seven HIV-1-positive individuals with low to moderate CD8 T cell responses, including five on highly active antiretroviral therapy (HAART). IFN-γ ELISPOT assays were performed using either monocytes or MDC to present antigens expressed by recombinant vaccinia viruses (r-VV). Methods Peripheral blood-derived monocytes were cultured for 5–6 days in the presence of IL-4 and granulocyte macrophage colony-stimulating factor, then matured in monocyte-conditioned medium. MDC were infected with r-VV and co-cultured in an ELISPOT assay with autologous monocyte-depleted PBMC. Results Relative to autologous monocytes, MDC amplified detection of antigen-specific CD8 T cells by 2–30-fold in response to antigens from HIV-1, Epstein–Barr virus and cytomegalovirus. Furthermore, antigenic specificities were revealed that had not been detected using standard ELISPOT of PBMC. Conclusion This assay will prove useful for the detection of memory T cells present at low frequency, and may be of interest for identifying subdominant cytotoxic T lymphocyte epitopes. This method may have broad applications for the detection of antiviral CD8 T cell responses in patient populations in whom such responses have been difficult to detect, including HIV-1-seropositive individuals with advanced disease or undergoing HAART.

The role of dendritic cells in the pathogenesis of HIV‐1 infection

Dendritic cells are professional antigen‐presenting cells required for generation of adaptive immunity. These cells are one of the initial target cells for HIV‐1 infection or capture of virions at site of transmission in the mucosa. DCs carrying HIV‐1 will migrate to the lymphoid tissue where they can contribute to the dissemination of the virus to adjacent CD4+ T cells. In addition, HIV‐1–exposed DCs may have impaired antigen‐presenting capacity resulting in inadequate expansion of HIV‐1–specific T cell responses. Here, we review the infection of different subtypes of DCs by HIV‐1 and the relevance of these cells in the transmission and establishment of HIV‐1 disease. In addition, we discuss the mechanisms through which HIV‐1–DC interactions could be exploited to optimise the generation and maintenance of HIV‐1–specific T cell immunity.

Cross-Presentation of Cell-Associated Antigens by Dendritic Cells

There is a strict requirement for professional antigen-presenting cells (APCs) in the generation of immunity toward most viruses. Exogenous pathways of MHC class I-restricted antigen presentation play an important role in the generation of antiviral immunity, particularly in the immune surveillance of virus-infected tissues of nonhematopoietic origin, and to bypass the detrimental effects of direct virus infection on professional APCs. The mechanisms underlying generation of antiviral immunity under these circumstances are discussed.