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ABSTRACT: Dendritic cells (DCs) are powerful activators of primary and secondary immune responses and have promising activity as anticancer vaccines. However, various populations of immune cells, including natural killer cells, regulatory T cells and especially cytotoxic T lymphocytes (CTLs), can inhibit DC function through cytotoxic clearance. Spontaneous tumor-specific CTL responses are frequently observed in patients before immunotherapy, and it is unclear how such pre-existing responses may affect DC vaccines. We used an adoptive transfer model to show that DC vaccination fail to induce the expansion of pre-existing CTLs or increase their production of interferon γ (IFNγ). The expansion and effector differentiation of naïve host CD8(+) T cells was also suppressed in the presence of CTLs of the same specificity. Suppression was caused by the cytotoxic functions of the adoptively transferred CTLs, as perforin-deficient CTLs could respond to DC vaccination by expanding and increasing IFNγ production. Proliferation and effector differentiation of host CD8(+) T cells as well as resistance to tumor challenge were also significantly increased. Expression of perforin by antitumor CTLs was critical in regulating the survival of vaccine DCs, while FAS/FASL and TRAIL/DR5 had a significant, but comparatively smaller, effect. We conclude that perforin-expressing CTLs can suppress the activity of DC-based vaccines and prevent the expansion of naïve and memory CD8(+) T cells as well as antitumor immune responses. We suggest that, paradoxically, temporarily blocking the cytotoxic functions of CTLs at the time of DC vaccination should result in improved vaccine efficiency and enhanced antitumor immunity.
Oncoimmunology. 12/2012; 1(9):1507-1516.
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ABSTRACT: 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.
The Journal of Immunology 01/2009; 181(12):8356-62. · 5.79 Impact Factor
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ABSTRACT: To examine the effects of route of administration and activation status on the ability of dendritic cells (DC) to accumulate in secondary lymphoid organs, and induce expansion of CD8(+) T cells and anti-tumor activity.
DC from bone marrow (BM) cultures were labeled with fluorochromes and injected s.c. or i.v. into naïve mice to monitor their survival and accumulation in vivo. Percentages of specific CD8(+) T cells in blood and delayed tumor growth were used as readouts of the immune response induced by DC immunization.
The route of DC administration was critical in determining the site of DC accumulation and time of DC persistence in vivo. DC injected s.c. accumulated in the draining lymph node, and DC injected i.v. in the spleen. DC appeared in the lymph node by 24 h after s.c. injection, their numbers peaked at 48 h and declined at 96 h. DC that had spontaneously matured in vitro were better able to migrate compared to immature DC. DC were found in the spleen at 3 h and 24 h after i.v. injection, but their numbers were low and declined by 48 h. Depending on the tumor cell line used, DC injected s.c. were as effective or more effective than DC injected i.v. at inducing anti-tumor responses. Pre-treatment with LPS increased DC accumulation in lymph nodes, but had no detectable effect on accumulation in the spleen. Pre-treatment with LPS also improved the ability of DC to induce CD8(+) T cell expansion and anti-tumor responses, regardless of the route of DC administration.
Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8(+) T cells in vivo.
Cancer Immunology and Immunotherapy 02/2008; 57(1):63-71. · 3.70 Impact Factor
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ABSTRACT: The survival of dendritic cells (DC) in vivo determines the duration of Ag presentation and is critical in determining the strength and magnitude of the resulting T cell response. We used a mouse model to show that Ag-loaded C57BL/6 DC (MHC class II(+/+) (MHC II(+/+))) that reach the lymph node survived longer than Ag-loaded MHC II(-/-) DC, with the numbers of C57BL/6 DC being approximately 2.5-fold the number of the MHC II(-/-) DC by day 4 and approximately 5-fold by day 7. The differential survival of DC in vivo was not affected by low doses of LPS, but in vitro pretreatment with CD40L or with high doses of LPS increased the numbers of MHC II(-/-) DC to levels approaching those of C57BL/6 DC. Regardless of their numbers and relative survival in lymph nodes, MHC II(-/-) DC were profoundly defective in their ability to induce CTL responses against the gp33 peptide epitope, and were unable to induce expansion and optimal cytotoxic activity of CD8(+) T cells specific for the male Ag UTY. We conclude that CD4(+) T cell help for CD8(+) responses involves mechanisms other than the increased survival of Ag-presenting DC in the lymph node.
The Journal of Immunology 12/2007; 179(9):5738-47. · 5.79 Impact Factor
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ABSTRACT: 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.
Leukemia and Lymphoma 05/2006; 47(4):675-82. · 2.58 Impact Factor
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ABSTRACT: The lifespan and survival of dendritic cells (DC) in vivo are potentially critical to the expansion of T cell immune responses. We have previously reported that DC loaded with specific antigen are rapidly eliminated by cytotoxic T lymphocytes (CTL) in vivo, but the site, mechanism, and consequences of DC elimination were not defined. In this article we show that DC elimination in vivo occurs in a perforin-dependent manner and does not require IFN-gamma or the presence of CD4(+)CD25(+) regulatory T cells. Most importantly, failure to eliminate DC had profound consequences on the CTL immune response. Perforin-deficient mice showed a progressive increase in the numbers of antigen-specific CD8(+) T cells after repeated immunizations with DC. In contrast, in control mice the number of antigen-specific CD8(+) T cells did not notably increase with repeated immunizations. Lastly, we also show that CTL-mediated elimination of DC occurs in peripheral tissues but not in the lymph node. Our data suggest that CTL act as "gatekeepers" that control access of antigen-loaded DC into the lymph node, thereby preventing continued expansion of antigen-specific T cells.
Proceedings of the National Academy of Sciences 02/2006; 103(1):147-52. · 9.68 Impact Factor
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ABSTRACT: Several reports have described a role of IL-4 in dendritic cell function. We have examined the number and phenotype of dendritic cells from C57Bl/6 wild-type and IL-4-/- mice, and compared their ability to induce T cell immune responses in vivo and in vitro. We observed that the number of dendritic cells in the spleens and lymph nodes of IL-4-/- mice is comparable to the number found in wild-type mice. In addition, the expression of maturation markers such as MHC II, CD40, CD80 and CD86, and of differentiation markers such as CD4, CD8 and CD11b, was also comparable in the two populations. Splenic wild-type and IL-4-/- dendritic cells were both able to present antigen to T cell receptor transgenic CD4+ or CD8+ T cells in culture. When pulsed with antigen in vitro and then injected subcutaneously into C57BL/6 host mice, both populations of dendritic cells were able to induce the division of T cell receptor transgenic CD4+ or CD8+ T cells in vivo. This was the case regardless of whether the antigen used in these experiments was a low or a high affinity T cell receptor ligand. Similarly, both populations of dendritic cells were able to activate antigen-specific cytotoxic T cell responses and initiate tumor-protective immune responses in vivo. We conclude that IL-4-/- and wild-type dendritic cells have a comparable ability to initiate T cell immune responses when in an IL-4-sufficient environment.
International Immunology 11/2004; 16(10):1451-8. · 3.41 Impact Factor
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ABSTRACT: Assessment of cell-mediated toxicity has traditionally been achieved by measuring the specific activity of enriched effector cell populations against antigen-loaded target cells labeled with radioactive isotopes in vitro. Fluorometric techniques are viewed as a promising alternative to the use of radioactive isotopes for these analyses. Direct assessment of cytotoxicity in vivo can be achieved by monitoring survival of injected fluorescent targets relative to a differentially labeled internal control population without specific antigen. We have developed this approach, incorporating the use of multiple target cell populations labeled with different dyes so that cytotoxicity can be assessed against titrated doses of a given antigen, or against a range of different antigens, simultaneously. We show that this assay, referred to as the VITAL assay, can be used to assess cytotoxic activity of CTL and iNKT cells in vivo and in vitro. CTL responses measured in vivo could be correlated with antigen doses used in immunization strategies, and also with the size of specific CTL populations enumerated in the blood with fluorescent MHC/peptide tetramers. The VITAL assay is, therefore, a sensitive technique allowing analysis of complex multi-epitope responses.
Journal of Immunological Methods 03/2004; 285(1):25-40. · 2.20 Impact Factor
