Preferential induction of CD4+ T cell responses through in vivo targeting of antigen to dendritic cell-associated C-type lectin-1

Targeting of Ags and therapeutics to dendritic cells (DCs) has immense potential for immunotherapy and vaccination. Because DCs are heterogeneous, optimal targeting strategies will require knowledge about functional specialization among DC subpopulations and identification of molecules for targeting appropriate DCs. We characterized the expression of a fungal recognition receptor, DC-associated C-type lectin-1 (Dectin-1), on mouse DC subpopulations and investigated the ability of an anti-Dectin-1 Ab to deliver Ag for the stimulation of immune responses. Dectin-1 was shown to be expressed on CD8alpha-CD4-CD11b+ DCs found in spleen and lymph nodes and dermal DCs present in skin and s.c. lymph nodes. Injection of Ag-anti-Dectin-1 conjugates induced CD4+ and CD8+ T cell and Ab responses at low doses where free Ag failed to elicit a response. Notably, qualitatively different immune responses were generated by targeting Ag to Dectin-1 vs CD205, a molecule expressed on CD8alpha+CD4-CD11b- DCs, dermal DCs, and Langerhans cells. Unlike anti-Dectin-1, anti-CD205 conjugates failed to elicit an Ab response. Moreover, when conjugates were injected i.v., anti-Dectin-1 stimulated a much stronger CD4+ T cell response and a much weaker CD8+ T cell response than anti-CD205. The results reveal Dectin-1 as a potential targeting molecule for immunization and have implications for the specialization of DC subpopulations.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Skin langerin+ dendritic cells transport intradermally injected anti-DEC-205 antibodies but are not essential for subsequent cytotoxic CD8+ T cell responses

Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.     

Comparison of the functional properties of murine dendritic cells generated in vivo with Flt3 ligand, GM-CSF and Flt3 ligand plus GM-SCF

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime na?ve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

Fc gamma receptor IIB on dendritic cells enforces peripheral tolerance by inhibiting effector T cell responses

The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c+ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the consequence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcgammaRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8+ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcgammaRIIB, resulting in a high incidence of diabetes. FcgammaRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcgammaRs. Further FcgammaRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcgammaRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcgammaRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcgammaRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of exogenously acquired Ag.     

Increased expression of mGITRL on D2SC/1 cells by particulate β-glucan impairs the suppressive effect of CD4+CD25+ regulatory T cells and enhances the effector T cell proliferation

β-Glucans have been shown to enhance immune responses for centuries, which contributes to their anti-tumor property. However, their mechanisms of action are still elusive. Dectin-1, the C-type lectin receptor for β-glucan, is expressed abundantly on dendritic cells (DCs). Activation of DCs via Dectin-1 can lead to the maturation of DC, inducing both innate and adaptive immune responses against tumor development and microbial infection. In this study, we found that particulate yeast-derived β-glucans could induce the maturation of murine dendritic cell line D2SC/1 cells and increase the expression of mGITRL on D2SC/1 cells via Dectin-1/Syk pathway in a dose dependent manner. Furthermore, we demonstrated that the increased mGITRL on D2SC/1 cells could impair the suppressive activity of CD4⁺CD25⁺ regulatory T cells (Tregs) and enhance the proliferation of CD4⁺CD25⁻ effector T cells (Teffs). These findings suggest that particulate β-glucan can be used as immunomodulator to stimulate potent T cell-mediated adaptive immunity while down-regulate immune suppressive activity, leading to a more efficient defense mechanism against tumor development or infectious diseases.     

Peptides identified through phage display direct immunogenic antigen to dendritic cells

Dendritic cells (DC) play a critical role in adaptive immunity by presenting Ag, thereby priming naive T cells. Specific DC-binding peptides were identified using a phage display peptide library. DC-peptides were fused to hepatitis C virus nonstructural protein 3 (NS3) while preserving DC targeting selectivity and Ag immunogenicity. The NS3-DC-peptide fusion protein was efficiently presented to CD4+ and CD8+ T cells derived from hepatitis C virus-positive blood cells, inducing their activation and proliferation. This immunogenic fusion protein was significantly more potent than NS3 control fusion protein or NS3 alone. In chimeric NOD-SCID mice transplanted with human cells, DC-targeted NS3 primed naive CD4+ and CD8+ T cells for potent NS3-specific proliferation and cytokine secretion. The capacity of peptides to specifically target immunogenic Ags to DC may establish a novel strategy for vaccine development.     

IFN-gamma inhibits presentation of a tumor/self peptide by CD8 alpha- dendritic cells via potentiation of the CD8 alpha+ subset

Using an in vivo model of tumor/self peptide presentation for induction of class I-restricted skin test reactivity, we have previously shown that a minority population of CD8+ dendritic cells (DC) negatively regulates the induction of T cell reactivity by peptide-loaded CD8- DC in DBA/2 mice. However, the CD8- fraction can be primed by IL-12 to overcome inhibition by the CD8+ subset when the two types of DC are cotransferred into recipient hosts. We report here that exposure of CD8+ DC to IFN-gamma greatly enhances their inhibitory activity on Ag presentation by the other subset, blocking the ability of IL-12-treated CD8- DC to overcome suppression. In contrast, IFN-gamma has no direct effects on the APC function of the latter cells and does not interfere with IL-12 signaling. The negative regulatory effect triggered by IFN-gamma in CD8+ DC appears to involve interference with tryptophan metabolism in vivo. Through tryptophan depletion affecting T cell responses, IFN-gamma acting on CD8+ DC may thus contribute to regulation of immunity to tumor/self peptides presented by the CD8- subset.     

Activation and route of administration both determine the ability of bone marrow-derived dendritic cells to accumulate in secondary lymphoid organs and prime CD8<Superscript>+</Superscript> T cells against tumors

Aims

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.

Methods

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.

Results

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.

Conclusions

Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8⁺ T cells in vivo.

     

Memory CD8+ T cell responses expand when antigen presentation overcomes T cell self-regulation

Antimicrobial memory CD8+ T cell responses are not readily expanded by either repeated infections or immunizations. This is a major obstacle to the development of T cell vaccines. Prime-boost immunization with heterologous microbes sharing the same CD8+ epitope can induce a large expansion of the CD8+ response; however, different vectors vary greatly in their ability to boost for reasons that are poorly understood. To investigate how efficient memory T cell expansion can occur, we evaluated immune regulatory events and Ag presentation after secondary immunization with strong and weak boosting vectors. We found that dendritic cells were essential for T cell boosting and that Ag presentation by these cells was regulated by cognate memory CD8+ T cells. When weak boosting vectors were used for secondary immunization, pre-established CD8+ T cells were able to effectively curtail Ag presentation, resulting in limited CD8+ T cell expansion. In contrast, a strong boosting vector, vaccinia virus, induced highly efficient Ag presentation that overcame regulation by cognate T cells and induced large numbers of memory CD8+ T cells to expand. Thus, efficient targeting of Ag to dendritic cells in the face of cognate immunity is an important requirement for T cell expansion.     

Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells

The activation of naive CD8+ T cells has been attributed to two mechanisms: cross-priming and direct priming. Cross-priming and direct priming differ in the source of Ag and in the cell that presents the Ag to the responding CD8+ T cells. In cross-priming, exogenous Ag is acquired by professional APCs, such as dendritic cells (DC), which process the Ag into peptides that are subsequently presented. In direct priming, the APCs, which may or may not be DC, synthesize and process the Ag and present it themselves to CD8+ T cells. In this study, we demonstrate that naive CD8+ T cells are activated by a third mechanism, called cross-dressing. In cross-dressing, DC directly acquire MHC class I-peptide complexes from dead, but not live, donor cells by a cell contact-mediated mechanism, and present the intact complexes to naive CD8+ T cells. Such DC are cross-dressed because they are wearing peptide-MHC complexes generated by other cells. CD8+ T cells activated by cross-dressing are restricted to the MHC class I genotype of the donor cells and are specific for peptides generated by the donor cells. In vivo studies demonstrate that optimal priming of CD8+ T cells requires both cross-priming and cross-dressing. Thus, cross-dressing may be an important mechanism by which DC prime naive CD8+ T cells and may explain how CD8+ T cells are primed to Ags that are inefficiently cross-presented.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Defective MHC class II presentation by dendritic cells limits CD4 T cell help for antitumor CD8 T cell responses

Cancer immunosurveillance failure is largely attributed to insufficient activation signals and dominant inhibitory stimuli for tumor Ag (TAg)-specific CD8 T cells. CD4 T cells have been shown to license dendritic cells (DC), thereby having the potential for converting CD8 T cell responses from tolerance to activation. To understand the potential cooperation of TAg-specific CD4 and CD8 T cells, we have characterized the responses of naive TCR transgenic CD8 and CD4 T cells to poorly immunogenic murine tumors. We found that whereas CD8 T cells sensed TAg and were tolerized, the CD4 T cells remained ignorant throughout tumor growth and did not provide help. This disparity in responses was due to normal TAg MHC class I cross-presentation by immature CD8alpha+ DC in the draining lymph node, but poor MHC class II presentation on all DC subsets due to selective inhibition by the tumor microenvironment. Thus, these results reveal a novel mechanism of cancer immunosubversion, in which inhibition of MHC-II TAg presentation on DC prevents CD4 T cell priming, thereby blocking any potential for licensing CD8alpha+ DC and helping tolerized CD8 T cells.     

Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses

Due to their capacity to induce primary immune responses, dendritic cells (DC) are attractive vectors for immunotherapy of cancer. Yet the targeting of tumor Ags to DC remains a challenge. Here we show that immature human monocyte-derived DC capture various killed tumor cells, including Jurkat T cell lymphoma, malignant melanoma, and prostate carcinoma. DC loaded with killed tumor cells induce MHC class I- and class II-restricted proliferation of autologous CD8+ and CD4+ T cells, demonstrating cross-presentation of tumor cell-derived Ags. Furthermore, tumor-loaded DC elicit expansion of CTL with cytotoxic activity against the tumor cells used for immunization. CTL elicited by DC loaded with the PC3 prostate carcinoma cell bodies kill another prostate carcinoma cell line, DU145, suggesting recognition of shared Ags. Finally, CTL elicited by DC loaded with killed LNCap prostate carcinoma cells, which express prostate specific Ag (PSA), are able to kill PSA peptide-pulsed T2 cells. This demonstrates that induced CTL activity is not only due to alloantigens, and that alloantigens do not prevent the activation of T cells specific for tumor-associated Ags. This approach opens the possibility of using allogeneic tumor cells as a source of tumor Ag for antitumor therapies.     

Treml4, an Ig superfamily member, mediates presentation of several antigens to T cells in vivo, including protective immunity to HER2 protein

Members of the triggering expressed on myeloid cells (Trem) receptor family fine-tune inflammatory responses. We previously identified one of these receptors, called Treml4, expressed mainly in the spleen, as well as at high levels by CD8α(+) dendritic cells and macrophages. Like other Trem family members, Treml4 has an Ig-like extracellular domain and a short cytoplasmic tail that associates with the adaptor DAP12. To follow up on our initial results that Treml4-Fc fusion proteins bind necrotic cells, we generated a knockout mouse to assess the role of Treml4 in the uptake and presentation of dying cells in vivo. Loss of Treml4 expression did not impair uptake of dying cells by CD8α(+) dendritic cells or cross-presentation of cell-associated Ag to CD8(+) T cells, suggesting overlapping function between Treml4 and other receptors in vivo. To further investigate Treml4 function, we took advantage of a newly generated mAb against Treml4 and engineered its H chain to express three different Ags (i.e., OVA, HIV GAGp24, and the extracellular domain of the breast cancer protein HER2). OVA directed to Treml4 was efficiently presented to CD8(+) and CD4(+) T cells in vivo. Anti-Treml4-GAGp24 mAbs, given along with a maturation stimulus, induced Th1 Ag-specific responses that were not observed in Treml4 knockout mice. Also, HER2 targeting using anti-Treml4 mAbs elicited combined CD4(+) and CD8(+) T cell immunity, and both T cells participated in resistance to a transplantable tumor. Therefore, Treml4 participates in Ag presentation in vivo, and targeting Ags with anti-Treml4 Abs enhances immunization of otherwise naive mice.     

Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect: dependence on the ADP-ribosyltransferase activity of the holotoxin

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c(+), CD8alpha(-), CD11b(-), B220(-), and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4(+) T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.     

Evaluation of DNA vaccination with recombinant adenoviruses using bioluminescence imaging of antigen expression: impact of application routes and delivery with dendritic cells

Recombinant DNA vaccines are able to induce strong CD8+ T cell mediated immunity and have become increasingly attractive for the prevention and treatment of infectious diseases and cancer. Dendritic cells (DC), which critically control cellular immune responses, have been transduced with antigen ex vivo and used as 'nature's adjuvant' to enhance vaccine efficacy. The impact of the application route on the in vivo distribution of antigen and the stimulation of CD8+ T cells have been subjects of considerable debate. Here we report the construction of vectors expressing a fusion protein between EGFP, the H2-K(b)-binding peptide OVA(aa257-264) and green click beetle luciferase as a model antigen which allows for simultaneous quantitative assessment of antigen expression using fluorescence and bioluminescence imaging in correlation with CD8+ T cell stimulation in vivo. We applied this construct to evaluate DNA vaccination with recombinant adenoviral vectors, assess the impact of using cultured DC for vaccine delivery and investigate different application routes. Antigen expression was non-invasively followed in vivo by visualizing bioluminescence with an ultrasensitive CCD camera. CD8+ T cell stimulation was detected with H2-K(b)-OVA(aa257-264) tetramers. We found that intravenous injection of adenovirus-transduced DC stimulated the strongest OVA(aa257-264)-specific cytotoxic T-lymphocyte (CTL) responses although it delivered two orders of magnitude less antigen in vivo when compared to direct injection of recombinant adenovirus. We believe that our experimental approach has the potential to facilitate translational development of improved genetic immunization strategies targeting DC directly in vivo.     

Characterization of chicken interleukin 2 receptor alpha chain, a homolog to mammalian CD25

To identify chicken IL-2R alpha chain (chCD25), the cDNA of chCD25 was cloned and mapped onto chicken chromosome 1. The polyclonal and monoclonal antibodies raised from the recombinant chCD25 specifically bound to the cell surface of splenic mononuclear cells (SMC) and inhibited chicken IL-2-dependent proliferation of T cells. Flow cytometry analysis revealed that chCD25 molecules could be expressed on the surface of monocytes/macrophages, thrombocytes, CD4+ and CD8+ cells as well as tissue cells. Importantly, the CD4+CD25+ and CD8+CD25+ cells were upregulated dramatically in chickens infected with H9N2 avian influenza virus. These results confirm that the cloned cDNA is the nucleotide sequence of chicken IL-2R, and suggest that chicken CD4+CD25+ and CD8+CD25+ cells may play an important role in immune responses induced by H9N2 virus, and the monoclonal antibodies to chCD25 may be useful for investigating biological functions of chicken regulatory T cells.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.