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Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

Denning WL, Xu J, Guo S, Klug CA, Hel Z - PLoS ONE (2011)

Bottom Line: Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model.Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting.Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Harnessing the ability of cytotoxic T lymphocytes (CTLs) to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs) following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

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Transplantation of genetically modified HSCs following nonmyeloablative conditioning results in a prolonged maintenance of antigen-specific CD8+ T cells.(A) B6 mice were immunized i.v. with 105 DCs from OVA-transgenic mice (DC-tOVA, left panel) or pretreated with BU (60 mg/ml) and two days later transplanted with 2×104 OVA-expressing HSCs (HSC-tOVA, right panel). Percentages of OVA-specific CD8+ T cells out of total CD8+ leukocytes were determined in blood of immunized animals at the indicated time points by staining with OVA-specific MHC-I pentamer and flow cytometry analysis. (B) Examples of flow cytometry analysis of OVA-specific CD8+ T cell immune responses in blood of two representative animals. (C) Mice were immunized with 500 or 2×104 DC-tOVA, immunodominant OVA peptide-coated DCs (DC-pOVA), or HSC-tOVA following BU pretreatment and the percentages of OVA-specific CD8+ T cells out of total CD8+ cells in blood were determined by OVA-specific MHC-I pentamer staining at indicated time points. Mice in the HSC-tOVA/RAD group were exposed to a split dose of 900 RADs prior to transplantation of 2×104 HSC-tOVA. 4 animals per group; error bars represent standard errors. Representative results of two of five similar experiments are presented.
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pone-0016897-g001: Transplantation of genetically modified HSCs following nonmyeloablative conditioning results in a prolonged maintenance of antigen-specific CD8+ T cells.(A) B6 mice were immunized i.v. with 105 DCs from OVA-transgenic mice (DC-tOVA, left panel) or pretreated with BU (60 mg/ml) and two days later transplanted with 2×104 OVA-expressing HSCs (HSC-tOVA, right panel). Percentages of OVA-specific CD8+ T cells out of total CD8+ leukocytes were determined in blood of immunized animals at the indicated time points by staining with OVA-specific MHC-I pentamer and flow cytometry analysis. (B) Examples of flow cytometry analysis of OVA-specific CD8+ T cell immune responses in blood of two representative animals. (C) Mice were immunized with 500 or 2×104 DC-tOVA, immunodominant OVA peptide-coated DCs (DC-pOVA), or HSC-tOVA following BU pretreatment and the percentages of OVA-specific CD8+ T cells out of total CD8+ cells in blood were determined by OVA-specific MHC-I pentamer staining at indicated time points. Mice in the HSC-tOVA/RAD group were exposed to a split dose of 900 RADs prior to transplantation of 2×104 HSC-tOVA. 4 animals per group; error bars represent standard errors. Representative results of two of five similar experiments are presented.

Mentions: To address whether sustained low-level expression of antigen results in an induction and maintenance of antigen-specific CTLs, recipient C57Bl/6 (B6) mice were transplanted with HSCs from OVA-transgenic donor mice on B6 background (HSC-tOVA). Two days prior transplantation, recipient animals were non-myeloablatively conditioned with busulfan (BU; 60 mg/kg i.v.) [23], [24]. HSC recipients were injected i.v. with a single dose of 500 or 2×104 of HSC-tOVA. Control groups were injected i.v. with 105 DCs derived from OVA-transgenic donors (DC-tOVA). The frequency of OVA-specific CD8+ T cells in DC-immunized mice peaked at 1 week after immunization and rapidly declined thereafter (Fig. 1A, left). In contrast, in HSC-tOVA-immunized mice, OVA-specific CD8+ T cells appeared with slower kinetics reaching high frequencies at weak 4 post transplantation (Fig. 1A, B). This is consistent with the appearance of circulating donor cells at 3–4 weeks post transplantation as evidenced by the occurrence of chimerism in CD45.2+ controls transplanted with CD45.1+ Lin− Sca-1+ HSCs (Fig. 2B and data not shown) [22]. Considerable variations in the frequencies of antigen-specific CD8+ T cells were observed among individual animals (Fig. 1A). A significant decrease in the frequency of antigen-specific T cells consistently occurred at 8 weeks post HSC transplantation followed by an increase at 12 weeks. This phenomenon is consistent with differences in the kinetics of appearance of cells descending from long-term self-renewing pluripotent HSCs versus short-term HSCs and partially differentiated lineage-committed common myeloid and lymphoid progenitors [22], [27]–[30]. The increase in the frequency of antigen-specific cells at 12 weeks suggests continuous low-level antigen expression. High levels of OVA-specific T cells were detectable at 16 weeks post vaccination but declined in most recipients at 20–24 weeks post treatment. Compared to transgenic DCs or DCs coated with a specific immunodominant peptide (DC-pOVA), administration of as few as 500 OVA-expressing HSCs into busulfan-pretreated animals resulted in a maintenance of significantly higher frequencies of OVA-specific cells at 4–16 weeks post immunization (Fig. 1C) (p<0.03 for HSC-tOVA versus DC-pOVA and HSC-tOVA versus DC-tOVA comparisons at 500 and 2×104 HSC vaccine doses at weeks 12 and 16). Pre-treatment of differentiated DC-tOVA or DC-pOVA with LPS (bacterial lipopolysaccharide, 100 ng/ml, 24 hrs) or administration of DCs subcutaneously into foot pad did not significantly enhance the longevity of induced immune responses determined as frequency of antigen-specific cells at weeks 4–16 post vaccination ([31] and data not shown). Importantly, mice transplanted following myeloablative conditioning (lethal irradiation, 900 RAD) with 2×104 HSCs displayed significantly lower levels of frequencies of antigen-specific CD8+ T cells throughout the observation period (Fig. 1C).


Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

Denning WL, Xu J, Guo S, Klug CA, Hel Z - PLoS ONE (2011)

Transplantation of genetically modified HSCs following nonmyeloablative conditioning results in a prolonged maintenance of antigen-specific CD8+ T cells.(A) B6 mice were immunized i.v. with 105 DCs from OVA-transgenic mice (DC-tOVA, left panel) or pretreated with BU (60 mg/ml) and two days later transplanted with 2×104 OVA-expressing HSCs (HSC-tOVA, right panel). Percentages of OVA-specific CD8+ T cells out of total CD8+ leukocytes were determined in blood of immunized animals at the indicated time points by staining with OVA-specific MHC-I pentamer and flow cytometry analysis. (B) Examples of flow cytometry analysis of OVA-specific CD8+ T cell immune responses in blood of two representative animals. (C) Mice were immunized with 500 or 2×104 DC-tOVA, immunodominant OVA peptide-coated DCs (DC-pOVA), or HSC-tOVA following BU pretreatment and the percentages of OVA-specific CD8+ T cells out of total CD8+ cells in blood were determined by OVA-specific MHC-I pentamer staining at indicated time points. Mice in the HSC-tOVA/RAD group were exposed to a split dose of 900 RADs prior to transplantation of 2×104 HSC-tOVA. 4 animals per group; error bars represent standard errors. Representative results of two of five similar experiments are presented.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040734&req=5

pone-0016897-g001: Transplantation of genetically modified HSCs following nonmyeloablative conditioning results in a prolonged maintenance of antigen-specific CD8+ T cells.(A) B6 mice were immunized i.v. with 105 DCs from OVA-transgenic mice (DC-tOVA, left panel) or pretreated with BU (60 mg/ml) and two days later transplanted with 2×104 OVA-expressing HSCs (HSC-tOVA, right panel). Percentages of OVA-specific CD8+ T cells out of total CD8+ leukocytes were determined in blood of immunized animals at the indicated time points by staining with OVA-specific MHC-I pentamer and flow cytometry analysis. (B) Examples of flow cytometry analysis of OVA-specific CD8+ T cell immune responses in blood of two representative animals. (C) Mice were immunized with 500 or 2×104 DC-tOVA, immunodominant OVA peptide-coated DCs (DC-pOVA), or HSC-tOVA following BU pretreatment and the percentages of OVA-specific CD8+ T cells out of total CD8+ cells in blood were determined by OVA-specific MHC-I pentamer staining at indicated time points. Mice in the HSC-tOVA/RAD group were exposed to a split dose of 900 RADs prior to transplantation of 2×104 HSC-tOVA. 4 animals per group; error bars represent standard errors. Representative results of two of five similar experiments are presented.
Mentions: To address whether sustained low-level expression of antigen results in an induction and maintenance of antigen-specific CTLs, recipient C57Bl/6 (B6) mice were transplanted with HSCs from OVA-transgenic donor mice on B6 background (HSC-tOVA). Two days prior transplantation, recipient animals were non-myeloablatively conditioned with busulfan (BU; 60 mg/kg i.v.) [23], [24]. HSC recipients were injected i.v. with a single dose of 500 or 2×104 of HSC-tOVA. Control groups were injected i.v. with 105 DCs derived from OVA-transgenic donors (DC-tOVA). The frequency of OVA-specific CD8+ T cells in DC-immunized mice peaked at 1 week after immunization and rapidly declined thereafter (Fig. 1A, left). In contrast, in HSC-tOVA-immunized mice, OVA-specific CD8+ T cells appeared with slower kinetics reaching high frequencies at weak 4 post transplantation (Fig. 1A, B). This is consistent with the appearance of circulating donor cells at 3–4 weeks post transplantation as evidenced by the occurrence of chimerism in CD45.2+ controls transplanted with CD45.1+ Lin− Sca-1+ HSCs (Fig. 2B and data not shown) [22]. Considerable variations in the frequencies of antigen-specific CD8+ T cells were observed among individual animals (Fig. 1A). A significant decrease in the frequency of antigen-specific T cells consistently occurred at 8 weeks post HSC transplantation followed by an increase at 12 weeks. This phenomenon is consistent with differences in the kinetics of appearance of cells descending from long-term self-renewing pluripotent HSCs versus short-term HSCs and partially differentiated lineage-committed common myeloid and lymphoid progenitors [22], [27]–[30]. The increase in the frequency of antigen-specific cells at 12 weeks suggests continuous low-level antigen expression. High levels of OVA-specific T cells were detectable at 16 weeks post vaccination but declined in most recipients at 20–24 weeks post treatment. Compared to transgenic DCs or DCs coated with a specific immunodominant peptide (DC-pOVA), administration of as few as 500 OVA-expressing HSCs into busulfan-pretreated animals resulted in a maintenance of significantly higher frequencies of OVA-specific cells at 4–16 weeks post immunization (Fig. 1C) (p<0.03 for HSC-tOVA versus DC-pOVA and HSC-tOVA versus DC-tOVA comparisons at 500 and 2×104 HSC vaccine doses at weeks 12 and 16). Pre-treatment of differentiated DC-tOVA or DC-pOVA with LPS (bacterial lipopolysaccharide, 100 ng/ml, 24 hrs) or administration of DCs subcutaneously into foot pad did not significantly enhance the longevity of induced immune responses determined as frequency of antigen-specific cells at weeks 4–16 post vaccination ([31] and data not shown). Importantly, mice transplanted following myeloablative conditioning (lethal irradiation, 900 RAD) with 2×104 HSCs displayed significantly lower levels of frequencies of antigen-specific CD8+ T cells throughout the observation period (Fig. 1C).

Bottom Line: Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model.Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting.Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

ABSTRACT
Harnessing the ability of cytotoxic T lymphocytes (CTLs) to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs) following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.

Show MeSH
Related in: MedlinePlus