Primary Cytotoxic T Lymphocytes Responses Research Articles

Dynamics of an HIV model with cytotoxic T-lymphocyte memory

We consider a four-dimensional HIV model that includes healthy cells, infected cells, primary cytotoxic T-lymphocyte response (CTLp), and secondary cytotoxic T-lymphocyte response (CTLe). The CTL memory generation depends on CD4+ T-cell help, and infection of CD4+ T cells results in impaired T-cell help. We show that the system has up to five equilibria. By the Routh–Hurwitz theorem and central manifold theorem we obtain some sufficient conditions for the local stability, globally stability of the equilibria, and the bifurcations. We still discover the bistability case where in the system there may coexist two stable equilibria or a stable equilibrium together with a stable limit cycle. Several numerical analyses are carried out to illustrate the validity of our theoretical results.

Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes

Several non-radioactive methods have widely been utilized to detect antigen-specific cytotoxic T lymphocyte (CTL) responses instead of the classical 51Cr-release assay. These methods include intracellular cytokine staining, major histocompatibility complex-class I tetramers, and the CD107a mobilization assay. However, they do not directly measure target-cell death. In contrast, several attempts have been made to develop the flow cytometric CTL (FC-CTL) assay for evaluation of cytotoxicity. However, further improvement is necessary for it to become standardized. Here, we evaluated the characteristics of the FC-CTL assay based on the uptake of propidium iodide (PI) using target cell lines expressing the green fluorescent protein (GFP). The FC-CTL assay was found to be sensitive enough to detect primary CTL responses. The usage of a pre-established GFP-expressing target cell line facilitated the procedure of the assay, and enabled a clear discrimination between target and effector cells. Time-course analyses demonstrated that PI-stained target cells were detected as early as surface CD107a expression after antigenic stimulation. Thus, the PI/GFP-based FC-CTL assay is sufficiently sensitive to practically detect the early stages of target-cell death, and may have a great potential for becoming a standard tool to measure CTL activity.

Toll-like receptor 9 is required for the maintenance of CD25+FoxP3+CD4+ Treg cells during Listeria monocytogenes infection

Abstract It has long been appreciated, but not understood, that the CD8+ cytotoxic T lymphocyte (CTL) dependence on CD4+T cell help (Th) is conditional; needed for some immunogens but not others. One explanation for this phenomenon envisions Th requirement as an intrinsic property of the pathogen itself rather than its introduction to the immune system. Here we show that dependence of the optimal CD8+ T cell response to Listeria monocytogenes (Lm) on CD4+ T cells is a function of the immunogen dose used for priming, with low dose Lm (LD; 50 or 103 CFU WT or ΔActA, respectively) inducing a primary antigen-specific CTL response profoundly dependent on CD4+ Th cells while that induced by high dose Lm (HD; 4×103 or 106 CFU WT or ΔActA, respectively) is significantly inhibited by CD4+CD25+FoxP3+regulatory T cells (Treg). The Th-independence of HD immunization is not overcome by additional antigen but instead involves the inflammatory response to more bacteria. Evaluation of various TLR pathways as the relevant sensing mechanism showed that TLR2 is required for CTL responses to LD immunization, and that HD immunization in the absence of TLR9 results in a simultaneous loss of CD25+FoxP3+CD4+ Treg cells and increase in conventional CD4+ Th cells and CTLs. Our data thus reveal that the CTL response to the same pathogen is determined by distinct roles for CD4+ T cells as helpers versus regulators based on immunogen dose and demonstrate a previously undescribed role for TLR9 in the regulation of CD4+ Th and Treg cells.

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

CD27 Agonism Plus PD-1 Blockade Recapitulates CD4+ T-cell Help in Therapeutic Anticancer Vaccination.

While showing promise, vaccination strategies to treat cancer require further optimization. Likely barriers to efficacy involve cancer-associated immunosuppression and peripheral tolerance, which limit the generation of effective vaccine-specific cytotoxic T lymphocytes (CTL). Because CD4(+) T cells improve CTL responsiveness, next-generation vaccines include helper epitopes. Here, we demonstrate in mice how CD4(+) T-cell help optimizes the CTL response to a clinically relevant DNA vaccine engineered to combat human papillomavirus-expressing tumors. Inclusion of tumor-unrelated helper epitopes greatly increased CTL priming, effector, and memory T-cell programming. CD4(+) T-cell help optimized the CTL response in all these aspects via CD27/CD70 costimulation. Notably, administration of an agonistic CD27 antibody could largely replace helper epitopes in promoting primary and memory CTL responses, acting directly on CD8(+) T cells. CD27 agonism improved efficacy of the vaccine without helper epitopes, more so than combined PD-1 and CTLA-4 blockade. Combining CD27 agonism with CTLA-4 blockade improved vaccine-induced CTL priming and tumor infiltration, but only combination with PD-1 blockade was effective at eradicating tumors, thereby fully recapitulating the effect of CD4(+) T-cell help on vaccine efficacy. PD-1 blockade alone did not affect CTL priming or tumor infiltration, so these results implied that it cooperated with CD4(+) T-cell help by alleviating immune suppression against CTL in the tumor. Helper epitope inclusion or CD27 agonism did not stimulate regulatory T cells, and vaccine efficacy was also improved by CD27 agonism in the presence of CD4(+) T-cell help. Our findings provide a preclinical rationale to apply CD27 agonist antibodies, either alone or combined with PD-1 blockade, to improve the therapeutic efficacy of cancer vaccines and immunotherapy generally. Cancer Res; 76(10); 2921-31. ©2016 AACR.

Sizing up the key determinants of the CD8+ T cell response

Naive CD8(+) T cells give rise to cytotoxic T lymphocytes (CTLs), which promote the effective eradication of viruses and tumours. Although the past decades have seen enormous advances in cellular immunology, a precise understanding of the key elements that determine the specificity and magnitude of primary CTL responses has been lacking. However, recent technological advances have allowed us to more accurately identify, characterize and quantitate key determinants that define the specificity and magnitude of CD8(+) T cell-mediated immunity. This Review discusses the technical and conceptual advances that have markedly changed our understanding of the determinants of primary CTL responses.

CD154+ CD4+ T-cell dependence for effective memory influenza virus-specific CD8+ T-cell responses.

CD40-CD154 (CD40 ligand) interactions are essential for the efficient priming of CD8(+) cytotoxic T lymphocyte (CTL) responses. This is typically via CD4(+)CD154(+) T-cell-dependent 'licensing' of CD40(+) dendritic cells (DCs); however, DCs infected with influenza A virus (IAV) upregulate CD154 expression, thus enabling efficient CTL priming in the absence of CD4(+) T activation. Therefore, it is unclear whether CD4 T cells and DCs have redundant or unique roles in the priming of primary and secondary CTL responses after infection. Here we determine the precise cellular interactions involved in CD40-CD154 regulation of both primary and secondary IAV-specific CTL responses. Infection of both CD40 KO and CD154 KO mice resulted in diminished quantitative and qualitative CTL responses after both primary and secondary infection. Adoptive transfer of CD154(+), but not CD154 KO, CD4 T cells into CD154 KO mice restored both primary and secondary IAV-specific CD8 T-cell responses. These data show that, although CD154 expression on CD4 T cells and other cell types (that is, DCs) may be redundant for the priming of primary CTL responses, CD154 expression by CD4 T cells is required for the priming memory CD8 T cells that are capable of fully responding to secondary infection.

A novel T cell-based vaccine capable of stimulating long-term functional CTL memory against B16 melanoma via CD40L signaling

The ultimate goal of antitumor vaccines is to develop memory CD8(+) cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. We previously demonstrated that the ovalbumin (OVA)-specific CD4(+) T cell-based (OVA-T(EXO)) vaccine generated using OVA-pulsed dendritic cell (DC(OVA))-released exosomes (EXO(OVA)) stimulate CTL responses via IL-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4(+) OVA-T(EXO) cells or OVA-T(EXO) cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2K(b)/OVA(257-264) tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10(OVA). We demonstrated that CD4(+) OVA-T(EXO) cells stimulated more efficient CTL responses compared to DC(OVA). By assessing primary and recall CTL responses in mice immunized with OVA-T(EXO) or with OVA-T(EXO) lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-T(EXO). Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10(OVA) tumors. Overall, this work suggests that a novel CD4(+) T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination.

CD154 and IL-2 Signaling of CD4+ T Cells Play a Critical Role in Multiple Phases of CD8+ CTL Responses Following Adenovirus Vaccination

Adenoviral (AdV) vectors represent most commonly utilized viral vaccines in clinical studies. While the role of CD8+ cytotoxic T lymphocyte (CTL) responses in mediating AdV-induced protection is well understood, the involvement of CD4+ T cell-provided signals in the development of functional CD8+ CTL responses remain unclear. To explore CD4+ T helper signals required for AdVova-stimulated CTL responses, we established an adoptive transfer system by transferring CD4+ T cells derived from various knock out and transgenic mice into wild-type and/or CD4-deficient animals, followed by immunizing with recombinant ovalbumin (OVA)-expressing AdVova vector. Without CD4+ T help, both primary and memory CTL responses were greatly reduced in this model, and were associated with increased PD-1 expression. The provision of OVA-specific CD4+ T help in CD4+ T cell-deficient mice restored AdVova-induced primary CTL responses, and supported survival and recall responses of AdVova-stimulated memory CTLs. These effects were specifically mediated by CD4+ T cell-produced IL-2 and CD154 signals. Adoptive transfer of “helped” or “unhelped” effector and memory CTLs into naïve CD4+ T cell-deficient or -sufficient mice also revealed an additional role for polyclonal CD4+ T cell environment in the survival of AdVova-stimulated CTLs, partially explaining the extension of CTL contraction phase. Finally, during recall responses, CD4+ T cell environment, particularly involving memory CD4+ T cells, greatly enhanced expansion of memory CTLs. Collectively, our data strongly suggest a critical role for CD4+ T help in multiple phases of AdV-stimulated CTL responses, and could partially explain certain failures in AdV-based immunization trials targeting malignant tumors and chronic diseases that are often associated with compromised CD4+ T cell population and function.

Blood-Stage Plasmodium berghei Infection Generates a Potent, Specific CD8+ T-Cell Response Despite Residence Largely in Cells Lacking MHC I Processing Machinery

Murine cerebral malaria is a complex disease caused by Plasmodium berghei ANKA infection. Several cell types, including CD8(+) T cells, are essential effectors of disease. Although the use of transgenic parasites expressing model antigens has revealed the induction of cytotoxic T lymphocytes (CTL) specific for these model antigens, there is no direct evidence for a response to authentic blood-stage parasite antigens, nor any knowledge of its magnitude. Our studies show that there is a dramatic primary parasite-specific CTL response, akin to viral immunity, reaching approximately 30% of splenic CD8(+) T cells, with many producing interferon-γ and tumor necrosis factor-α. These cells express granzyme B and other markers of specific responders, are cytolytic, and respond to a broad array of major histocompatibility complex (MHC) I-restricted epitopes, 5 of which are identified here. Our studies indicate that vigorous CTL responses can be induced to pathogens even when they largely reside in red blood cells, which lack MHC I processing machinery.

Prospects for T cell immunotherapy of tumours by vaccination with immunodominant and subdominant peptides.

Immunotherapy of tumours by adoptive transfer of cytotoxic T lymphocytes (CTL) is now feasible in experimental murine systems. These CTL recognize peptide sequences of defined length presented by major histocompatibility complex (MHC) class I molecules. Effective eradication of large tumour masses requires co-administration of interleukin 2. Tumour escape strategies are numerous but in various instances can be counteracted by defined measures. Initiation of CTL responses against poorly immunogenic virally induced tumours and other tumours requires novel strategies to overcome T cell inertia. We propose a strategy in which CTL are raised against target molecules of choice including differentiation antigens of restricted tissue distribution (autoantigens) or mutated/overexpressed oncogene products. The steps proposed include: (1) identification of target molecules of choice. (2) Identification in these target molecules of peptides fitting MHC allele-specific peptide motifs involved in peptide binding to MHC molecules. (3) Evaluation of actual binding of such peptides to specific MHC class I molecules. (4) In vitro CTL response induction by such peptides, presented by highly efficient antigen-presenting cells such as antigen processing-defective cells carrying empty MHC class I molecules loaded with a single peptide or dendritic cells. Both types of cells are capable of primary CTL response induction in vitro. (5) Evaluation of proper processing by the demonstration of tumour cell lysis by these CTL. (6) Adoptive transfer of tumour-specific CTL generated in vitro or vaccination with peptides. These various steps have now been taken for several viruses, virally induced tumours and other types of tumours and the first indications that this strategy is useful have been obtained.

Lentiviral Vectors Encoding HIV-1 Polyepitopes Induce Broad CTL Responses In Vivo

Lentiviral Vectors Encoding HIV-1 Polyepitopes Induce Broad CTL Responses In Vivo

Early acquisition of cytolytic function and transcriptional changes in a primary CD8+ T-cell response in vivo

AbstractFunctional studies show that programming of CD8+ T cells occurs early after initial antigen encounter within as little as 2 hours. To define the molecular basis of these events, we transferred TCR transgenic T cells from F5 Rag−/− mice into naive recipients and stimulated them with recombinant vaccinia expressing the immunodominant influenza epitope NP366-374. Transcription in epitope-specific cytotoxic T lymphocytes (CTLs) was analyzed using Affymetrix 430 2.0 GeneChips and quantitative polymerase chain reaction (PCR). We demonstrated an early transcriptional burst with the greatest number of genes reaching peak expression 12 hours after stimulation. Using in vivo cytotoxicity assays we demonstrated that early up-regulation of cytolytic genes was accompanied by acquisition of killing capacity within 24 hours of stimulation. However, T-cell proliferation was not observed until 48 hours. We therefore conclude that clonal expansion rather than acquisition of effector function is the rate-limiting step in the development of a primary CTL response.

Generation of tumoricidal PAX3 peptide antigen specific cytotoxic T lymphocytes

The transcription factor PAX3 is expressed during early embryogenesis and in multiple cancer types, including embryonal rhabdomyosarcoma (ERMS), Ewing sarcoma (ES) and malignant melanoma (MEL), suggesting that it could function as a general tumor associated antigen. Major histocompatibility complex (MHC) peptide binding algorithms were used to predict potential epitopes in PAX3 capable of stimulating in vitro naïve HLA-A0201 restricted cytotoxic T-lymphocytes (CTLs). Two peptides, PAX3-282 (QLMAFNHLI) and a modified version of this peptide PAX3-282.9V (QLMAFNHLV), were capable of inducing antigen-specific CTLs. Of these peptides, PAX3-282.9V was the most efficient inducer of primary CTL response. These CTLs were able to lyse HLA-A0201 expressing target cells that were pulsed with peptide, and more importantly, were effective in killing tumor cells that express PAX3, including ERMS, ES and MEL cell lines. These findings provide compelling evidence that peptide PAX3-282 is naturally processed by tumors and is presented in the context of HLA-A0201 in adequate amounts to allow CTL recognition. Also, PAX3-282.9V is an effective immunogenic peptide able to induce CTL recognition of PAX3-containing tumors and may be used as an antitumor peptide vaccine.

Induction of effective therapeutic antitumor immunity by direct in vivo administration of lentiviral vectors

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

In Vivo Depletion of CD4+CD25+ Regulatory T Cells Enhances the Antigen-Specific Primary and Memory CTL Response Elicited by Mature mRNA-Electroporated Dendritic Cells

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). Here, we further optimize the protocol and evaluate the capacity of mRNA-electroporated DC as a vaccine for immunotherapy. First, the early DC maturation kinetics and the effect of different lipopolysaccharide incubation periods on the phenotypic maturation profile of DC are determined. Next, we show that either immature or mature DC are equally well electroporated and express and present the transgene at a comparable level after electroporation. We point out that the mRNA electroporation results in a negative effect on the interleukin (IL)-12p70, IL-6, and tumor necrosis factor-α secretion after maturation. Nevertheless, mRNA-electroporated DC induce an effective cytotoxic T lymphocyte (CTL) response in vivo. Mature electroporated DC are significantly more potent in eliciting an Ag-specific CD8+ CTL response compared to their immature electroporated counterparts. In addition, a significant improvement in CTL response is obtained both in the primary and in the memory effector phases when CD4+CD25+ regulatory T cells (Treg) are depleted in vivo prior to immunization. These findings are further substantiated in tumor protection experiments and hold convincing evidence for the merit of Treg cell depletion prior to immunization with mRNA-electroporated DC.

Interleukin-12 Increases Proliferation and Interferon-γ Production but Not Cytolytic Activity of Human Antigen-Specific Effector Memory Cytotoxic T Lymphocytes: Power of the Effect Depends on the Functional Avidity of the T Cell and the Antigen Concentration

Cytotoxic T lymphocytes (CTLs) play an important role in the defense against viral infections and malignant diseases. Interleukin (IL)-12 plays a crucial role in induction of antigen-specific primary CTL responses and enhances proliferation, interferon-γ (IFN-γ) production, and cytolytic activity of mitogen-stimulated T cells. However, the effects of IL-12 on proliferation and effector functions of previously in vitro or in vivo primed antigen-specific CTLs are less clear. Our results show that IL-12 induces an increase in proliferation of and IFN-γ production by influenza peptide-specific CTLs, but no increase in cytolytic activity on a per cell basis was observed in bulk cultures. Stimulation of a CTL clone confirmed these results; IL-12 supported an increase in IFN-γ production, but did not increase cytolytic activity. The extent of the effect of IL-12 on IFN-γ production differs per CTL clone and depends on the avidity of the clone and the peptide concentration on its target. Our data suggest that IL-12 is a good adjuvant for boosting CTL responses, in terms of proliferation and IFN-γ production, the latter particularly for CTLs with low to intermediate avidity, such as tumor-associated self-antigen-specific CTLs.

Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8(+) cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8(+) CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.

Cotransfection of dendritic cells with RNA coding for HER-2/neu and 4-1BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes

Ribonucleic acid (RNA) transfection of dendritic cells (DCs) was shown to be highly efficient in eliciting CD8+ and CD4+ T-cell responses. We analyzed whether electroporation of DCs with RNA coding for a tumor-associated antigen (TAA) would elicit antigen-specific effector cytotoxic T lymphocyte (CTL) responses and whether these responses could be modulated by cotransfection with a second specific synthetic RNA. Therefore in vitro generated human monocyte-derived DCs were electroporated with in vitro transcribed RNA (in vitro transcript, IVT) encoding the TAA HER-2/neu. Additionally, these cells were cotransfected with IVT coding for human 4-1BBL. Transfection of DCs with 4-1BBL-IVT did not alter their typical phenotype. However, it increased the expression of the costimulatory molecules CD80 and CD40. Coadministration of HER-2/neu- and 4-1BBL-IVT resulted in an increased specific lysis of target cells by the in vitro induced CTL lines, indicating that 4-1BBL enhances their ability to elicit primary CTL responses. Interestingly, transfection of DCs with 4-1BBL-IVT did not augment their capacity to stimulate allogeneic lymphocyte responses. The here established approach of cotransfection of DCs with tumor-RNA and a second specific IVT could improve and optimize the in vitro manipulation of DCs for the induction of antigen-specific CTL responses.

991. Adenovirus-Mediated IL-12 Gene Transfer Increase Virus Specific CD8 T Cell Response in Aged Mice

Top of pageAbstract Respiratory viral infection is a major cause of morbidity and mortality in the elderly population. IL-12 and other Th1 cytokines can prevent the age-related defect in the cytotoxic T lymphocyte (CTL) response by blocking the age-associated down modulation of CD28 and immune senescence of CD8+ T cells. Gene therapy is one method to restore defective Th1 cytokines and reverse immune senescence. However, the effect of IL-12 on the generation and effector function of the viral epitope-specific cytotoxic CD8 T cells (CTLs) after delivery by an adenovirus vector has been difficult to dissect due to the lack of appropriate tetramers capable of identifying the virus-specific CTL response. This limitation can now be overcome by the use of our recently developed MHC class I tetramer containing the adenovirus (Ad) epitope peptide E1Bp, referred to as Db-E1Bp, in combination with the use of an in vivo killing assay to analyze the virus specific CD8 T cell response in young and old mice after Ad administration. To determine the effect of IL-12 on the generation and effector function of the virus-specific CTLs, we utilized an recombinant Ad vector to deliver the IL-12 gene into aged (16-mo-old) and young (2-mo-old) C57BL/6 (B6; H-2b) mice before infected with a wild-type Ad5. The primary CTL response was assayed by the Db-E1Bp tetramer and E1Bp-specific in vivo killing assay. Our results indicate that there was a 58% decrease in the production of virus-specific Db-E1Bp+CD8+ T cells in aged mice at day 8 after Ad5 infection, compared with that from young mice (p<0.01; Table). This was associated with a 30% decrease in the specific killing activity by these CTLs in aged mice (p<0.01). In vivo pretreatment of aged mice with Ad-IL12 resulted in an increase in serum IL-12, from 18|[plusmn]|7 ng/ml before pretreatment to 96|[plusmn]|12 ng/ml at the time of Ad5 infection 3 days later. Ad-IL12 pretreatment also induced strong IFNg production of 34|[plusmn]|11 ng/ml at the time of Ad5 injection compared to the base line of 0.5|[plusmn]|0.2 ng/ml. There was only a moderate increase of these cytokines in young mice. However, the serum IL-12 correlated with IFNg production in both old (r2=0.889; p<0.001) and young (r2=0.946; p<0.001) mice. At day 8 after Ad5 infection, both the Db-E1Bp+CD8+ /total CD8+ T cells and E1Bp specific lysis efficiency in the spleen and liver of aged mice increased to levels comparable to those of young mice (Table). This was associated with an increased thymocyte proliferative response mediated through IL-2 and IL-7 in aged mice. Our results indicate that aged mice have a defect in the CTL response to Ad infection, and that Ad-IL12 treatment significantly enhances the Ad-specific CTL response in aged mice, partially by induction of IFN|[gamma]|.