Stimulation Of Cytotoxic T Lymphocytes Research Articles

Lyapunov functionals for a general time-delayed virus dynamic model with different CTL responses.

A time-delayed virus dynamic model is proposed with general monotonic incidence, different nonlinear CTL (cytotoxic T lymphocyte) responses [CTL elimination function pyg1(z) and CTL stimulation function cyg2(z)], and immune impairment. Indeed, the different CTL responses pose challenges in obtaining the dissipativeness of the model. By constructing appropriate Lyapunov functionals with some detailed analysis techniques, the global stability results of all equilibria of the model are obtained. By the way, we point out that the partial derivative fv(x,0) is increasing (but not necessarily strictly) in x>0 for the general monotonic incidence f(x,v). However, some papers defaulted that the partial derivative was strictly increasing. Our main results show that if the basic reproduction number R0≤1, the infection-free equilibrium E0 is globally asymptotically stable (GAS); if CTL stimulation function cyg2(z)=0 for z=0 and the CTL threshold parameter R1≤1<R0, then the immunity-inactivated infection equilibrium E1 is GAS; if the immunity-activated infection equilibrium E+ exists, then it is GAS. Two specific examples are provided to illustrate the applicability of the main results. The main results acquired in this paper improve or extend some of the existing results.

Designing of neoepitopes based vaccine against breast cancer using integrated immuno and bioinformatics approach

Cancer is characterized by genetic instability due to accumulation of somatic mutations in the genes which generate neoepitopes (mutated epitopes) for targeting by Cytotoxic T lymphocytes (CTL). Breast cancer has a high transformation rate with unique composition of mutational burden and neoepitopes load that open a platform to designing a neoepitopes-based vaccine. Neoepitopes-based therapeutic cancer vaccines designed by neoantigens have shown to be feasible, nontoxic, and immunogenic in cancer patients. Stimulation of CTL by neoepitope-based vaccine of self-antigenic proteins plays a key role in distinguishing cancer cells from normal cells and selectively targets only malignant cells. A neoepitopes-based vaccine to combat breast cancer was designed by combining immunology and bioinformatics approaches. The vaccine construct was assembled by the fusion of CTL neoepitopes, helper sequences (used for better separation of the epitopes), and adjuvant together with linkers. The neoepitopes were identified from somatic mutations in the MUC16, TP53, RYR2, F5, DNAH17, ASPM, and ABCA13 self-antigenic proteins. The vaccine construct was undertaken to study the immune simulations (IS), physiochemical characteristics (PP), molecular docking (MD) and simulations, and cloning in appropriate vector. Together, these parameters establish safety, stability, and a strong binding affinity against class I MHC molecules capable of inducing a complete immune response against breast cancer cells. Communicated by Ramaswamy H. Sarma

Characterization of the Class I MHC Peptidome Resulting From DNCB Exposure of HaCaT Cells.

Skin sensitization following the covalent modification of proteins by low molecular weight chemicals (haptenation) is mediated by cytotoxic T lymphocyte (CTL) recognition of human leukocyte antigen (HLA) molecules presented on the surface of almost all nucleated cells. There exist 3 nonmutually exclusive hypotheses for how haptens mediate CTL recognition: direct stimulation by haptenated peptides, hapten modification of HLA leading to an altered HLA-peptide repertoire, or a hapten altered proteome leading to an altered HLA-peptide repertoire. To shed light on the mechanism underpinning skin sensitization, we set out to utilize proteomic analysis of keratinocyte presented antigens following exposure to 2,4-dinitrochlorobenzene (DNCB). We show that the following DNCB exposure, cultured keratinocytes present cysteine haptenated (dinitrophenylated) peptides in multiple HLA molecules. In addition, we find that one of the DNCB modified peptides derives from the active site of cytosolic glutathione-S transferase-. These results support the current view that a key mechanism of skin sensitization is stimulation of CTLs by haptenated peptides. Data are available via ProteomeXchange with identifier PXD021373.

Abstract 3226: The IRF8-osteopontin-CD44 axis functions as an immune checkpoint to control CD8+ T cell activation and tumor immune evasion

Abstract Despite breakthroughs in immune checkpoint inhibitor (ICI) immunotherapy, not all human cancers respond to ICI immunotherapy and only fraction of patients with responsive tumors have a durable response to current ICI immunotherapy. This clinical conundrum suggests that additional immune checkpoints may exist, particularly in cancers resistant to current ICI immunotherapy, such as colorectal cancer. We report here that interferon regulatory factor 8 (IRF8) deficiency led to impairment of cytotoxic T lymphocyte (CTL) activation in a peptide vaccine model and allowed allograft transplant tumor tolerance. These effects were associated with upregulation of the CTL surface marker CD44. However, analysis of chimeric mice with competitive reconstitution of wild type and IRF8 KO bone marrow cells as well as mice with IRF8 deficiency only in T cells indicated that IRF8 plays no intrinsic role in CTL activation. Instead, IRF8 functioned as a repressor of osteopontin (OPN), the physiological ligand for CD44 on T cells, in CD11b+Ly6CloLy6G+ myeloid cells and OPN acted as a potent T cell suppressor. In vitro stimulation of CTLs in the presence of OPN resulted in decreased expression of activation markers CD69 and CD25 and inhibited proliferation and interferon gamma (IFNg) secretion. Expression of OPN was found to be upregulated in both myeloid cells and colon epithelial cells following silencing of IRF8 expression. IRF8 bound to the Spp1 promoter, which encodes OPN, to repress OPN expression in colon epithelial cells. Correspondingly, human colon carcinoma cells exhibited decreased IRF8 and increased OPN expression. These increased OPN levels inhibited human PBMC proliferation and IFNg secretion. The elevated expression of OPN in human colon carcinoma was correlated with decreased patient survival. Our data indicates that myeloid and tumor cell-expressed OPN acts as a novel immune checkpoint to suppress T cell activation and confer host tumor immune tolerance. Blockade of this checkpoint may expand the pool of patients who may benefit from ICI immunotherapy. Citation Format: John D. Klement, Amy V. Paschall, Priscilla S. Redd, Mohammed L. Ibrahim, Chunwan Lu, Dafeng Yang, Esteban Celis, Scott I. Abrams, Keiko Ozato, Kebin Liu. The IRF8-osteopontin-CD44 axis functions as an immune checkpoint to control CD8+ T cell activation and tumor immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3226.

Repression of osteopontin by IRF8 regulates a novel immunosuppressive checkpoint

Abstract While current immune checkpoint inhibitors (ICIs) have shown efficacy in a wide variety of human cancers, many patients and cancer types fail to respond to ICI immunotherapy, suggesting the presence of other immunoinhibitory checkpoints. We report here that a deficiency in the transcription factor interferon regulatory factor 8 (IRF8) led to a loss of function of cytotoxic T lymphocytes (CTLs) in both an allograft tumor and a peptide vaccine model. However, a T cell-specific knockout and a mixed chimera model demonstrated that IRF8 suppressed CTL activation through a CTL-extrinsic mechanism. This was found to be through IRF8-dependent suppression of the matricellular protein osteopontin (OPN). During colorectal cancer (CRC) development, IRF8 is downregulated in both tumor-associated CD11b+Ly6CloLy6Ghi myeloid cells and colon epithelial cells, where it represses OPN expression by binding to its promoter. Accordingly, in both mouse CRC models and human CRC patients, transcriptional downregulation of IRF8 was accompanied by an upregulation of OPN at the transcriptional and protein level. Enhanced levels of OPN were found to negatively correlate with patient survival. In vitro stimulation of mouse and human CTLs in the presence of OPN led to decreased activation and interferon gamma secretion. Similarly, deletion of spp1, which encodes for OPN, from murine CT26 colon adenocarcinoma cells caused increased susceptibility to CTL-mediated lysis and enhanced CTL activation. As such, tumor and myeloid-derived OPN may function as a novel immune checkpoint to restrain host CTL activation.

IL-12 stimulates CTLs to secrete exosomes capable of activating bystander CD8+ T cells

An effective cytotoxic T lymphocyte (CTL) response against intracellular pathogens is generally accomplished by immense CTL expansion and activation, which can destroy infected cells. Vigorous immune responses can lead to activation of bystander CD8+ T cells, but the contribution from antigen-specific CTLs is not well understood. We found that CTLs secrete extracellular vesicles following antigen stimulation. These CTL-derived vesicles contain CTL proteins and exhibit markers and size profiles consistent with exosomes. Interestingly, further stimulation of CTLs with IL-12 impacts exosome size and leads to selective enrichment of certain exosomal proteins. More important, exosomes from IL-12-stimulated CTLs directly activated bystander naïve CD8+ T cells to produce interferon-γ (IFNγ) and granzyme B (GZB) in the absence of antigens, whereas control exosomes derived from antigen-stimulated CTLs did not. In addition, IL-12 induced exosomes are able to strengthen the effects of weak antigen stimulation on CTLs. Proteomic analysis demonstrates that IL-12 stimulation alters catalytic and binding activities of proteins in CTL exosomes. Our findings indicate that the biological function and morphology of exosomes secreted by CTLs can be influenced by the type of stimulation CTLs receive. Thus, a fully functional, ongoing, antigen-specific CTL response may influence bystander CD8+ T cells through secretion of exosomes.

Development and immunological evaluation of HLA-specific chronic myeloid leukemia polyepitope vaccine in Chinese population

BackgroundBCR/ABL and Wilms’ tumor 1 (WT1) are an ideal tumor associated antigens which can be used to develop a potential chronic myeloid leukemia (CML) dentritic cell (DC) vaccine. Here, we constructed a novel polyepitope vaccine which used recombinant lentiviral vector carrying BCR/ABL and WT1 genes, and determined the immunological effects of this vaccine in vitro. MethodsThe DC vaccine was constructed using lentiviral vector transduced DCs. T lymphocytes were stimulated with DC vaccine and then co-cultured in vitro with peripheral blood mononuclear cells (PBMCs) from CML or ALL patients, respectively. The cytotoxicity of proliferous cytotoxic T lymphocytes (CTLs) was determined by the LDH assay. The IFN-γ production of CTLs was detected using ELISPOT assay. ResultsWe constructed an lentiviral vector encoding 50 different epitopes from BCR/ABL and WT1 antigens, and transferred it into DCs to prepare the DC vaccine successfully. The in vivo stimulation of CTLs with this DC vaccine were proved to show strong cytotoxicity and produce high level of IFN-γ. ConclusionsThe novel recombinant lentiviral polyepitope DC vaccine is a promising candidate for clinical trials and may be an effective approach for CML immunotherapy.

Screening For Anti-Leukemic CTLs After Allogeneic Hematopoietic Stem Cell Transplantation Applying Streptamer Technology

IntroductionGreiner et al. demonstrated data suggesting a promising link between CD8+ T cell responses with a specificity of NPM1mut and graft versus leukemia effect (GvL). In patients with acute myeloid leukemia (AML) who underwent hematopoietic stem cell (HSC) transplantation, effector cells of the newly established immune system are thought to elicit a potent graft versus leukemia (GvL) effect eradicating residual leukemic cells. Cytotoxic T-lymphocytes (CTLs) specific for leukemia associated MHC-presented peptides may play a crucial role in the anti-tumor reaction and in achieving complete molecular remission. Leukemic cells are detectable after allogeneic HSCT in AML at least at the level of minimal residual disease (MRD). Malignant cells could therefore be identified and targeted by effector cells of the new immune system and stimulation of CTLs by antigen is assured. Detectable leukemia-specific CTLs will allow studies which address the correlation of the course of CTLs and clinical events. Further, it could allow for an assessment of the functional properties of these cells with respect to GvL effects. MethodsWe screened HLA-A0201 positive patients with AML and chronic lymphocytic leukemia (CLL) biweekly from day 28 after transplantation until day 112 for antileukemic CTLs applying streptamer technology. For AML a panel of 7 MHC peptide complexes was used to label CTLs from peripheral blood: phycoerythrin-(PE)-labeled streptamers refolded with peptides (number of different peptides) derived from the amino acid sequence of the proteins WT1 (1), PR3 (1), RHAMM (1), mutated NPM1 (2), and survivin (2). For CLL peptides derived from RHAMM (1), survivin (2) and fibromodulin (4) were chosen for screening. Peptides from cytomegalovirus (CMV) protein pp65 and from Influenza matrix protein M1 were used as positive controls. A peptide of the HIV reverse transcriptase served as negative control. ResultsUp to now neither for AML (n = 9) nor for CLL (n = 1), leukemia specific CTLs were detected according to the respective panel at any time point. However, it was possible to detect CMV-specific CTLs in most of the CMV seropositive patient-recipient pairs and frequencies increased when CMV reactivation occurred. Increasing frequencies of CMV-specific CTLs were even measured in CMV seropositive patients who received allogeneic HSC grafts from CMV seronegative donors, proving the adequacy of the method and reflecting the stimulation and proliferation of CMV-specific CTLs. DiscussionDifferent reasons may account for our negative results so far: the limited numbers of selected peptides, which may not be immunodominant or the small number of screened patients are two of them. It must be considered that the CTL immune response to virus infection and concomitant CMV clearance may not represent a proper model for the GvL effect, which possibly is characterized only by a weak proliferation of CTLs resulting in cell counts below the detection limit of the applied flowcytometric measurements. We will further extend our screening approach to a minimum of 18 individuals per specificity. If frequencies above the limit of detection will not be detected in at least one out of 18 patients the 95 % confidence interval of the true percentage of patients with measurable CTLs of this specificity should be < 15 %. ConclusionsThis study highlights the difficulties to study leukemia-specific CTLs. So far, leukemia-specific CTLs have only been measured in single patients after allogeneic HSCT. Information on cohorts of patients who have been monitored longitudinally for specific CTLs is very rare. The kinetics of leukemia-specific CTLs which mediate GvL effects are almost unknown. Further research on delineating T-cell mediated GvL-effects and realistic estimates for frequencies of specific CTLs at well defined time-points are urgently needed. Disclosures:No relevant conflicts of interest to declare.

Cord Blood Allograft Rejection Mediated By Coordinated Donor-Specific Cellular and Humoral Immune Processes

Recent statistical analyses of the clinical outcome have revealed that the presence of donor-specific anti-HLA antibodies (DSAs) in pre-transplant recipient is correlated with increased risk for graft rejection after allogeneic hematopoietic stem cell (HSC) transplantation. However, while cytotoxic T lymphocytes (CTLs) recognizing mismatched HLA molecule of the donor have been shown to be involved in graft rejection, there has been no biological evidence in humans that graft rejection is mediated by DSAs. In the present study, we demonstrate a case of cord blood allograft rejection in which DSAs acted to mediate graft rejection. Interestingly, CTL clones specific for donor HLA molecule were also isolated, suggesting that humoral and cellular immune responses were together responsible for allograft rejection. A patient with graft rejection after HLA-mismatched cord blood transplantation was studied. The white blood cell count increased transiently, subsequently decreased to undetectable level, and finally graft rejection was diagnosed on day 34. Screening for pre-transplant anti-HLA antibodies was not routinely performed at that time. We initially presumed that DSA would be responsible for graft rejection. The patient serum on day 36 were screened for HLA antibodies and further evaluated to determine HLA specificities using a LABScreen Single Antigen Kit. An antibody against HLA-B* 54:01, which was expressed in donor cells but not in patient cells, was detected. To test the effect of the DSA against HLA-B* 54:01 on HSC engraftment, antibody-dependent cellular cytotoxicity (ADCC) activity was assessed using unrelated HLA-B* 54:01-positive bone marrow mononuclear cells (BMMNCs) pre-cultured with patient serum and pre-transplant NK cells. The patient serum clearly showed an inhibitory effect on colony formation. Complement-dependent cytotoxicity activity of the DSA could not be detected. These data suggest that the DSA impaired the cord blood engraftment through ADCC activities. We next determined if cellular immunity was also responsible for allograft rejection. Two independent CTL clones, CD4-CD8+ and CD4+CD8-, were isolated from the patient blood at the time of graft rejection by limiting dilution. Both CTL clones were of patient origin by using short tandem repeat analysis and showed cytotoxicity against donor cells but not patient cells in Cr release assay. A CTL stimulation assay using COS cells transfected with various mismatched donor HLA cDNA constructs demonstrated that CD8+ and CD4+CTL clones recognized HLA-B* 54:01 and -DRB1* 15:02 molecules, respectively, both of which were expressed in donor cells but not in patient cells. To test the effect of the CTL clones on engraftment, a colony forming assay using either HLA-B* 54:01 or -DRB1* 15:02-positive unrelated BMMNCs pre-cultured with the corresponding CTL clones was performed. Each of these CTL clones inhibited colony formation from unrelated BMMNCs sharing target HLA in a dose-dependent manner. These data suggest that the CTLs also impaired the cord blood engraftment. Furthermore, anti-HLA-B* 54:01 antibody was detected in cryopreserved pre-transplant patient serum, and in nested PCR assays specific for the HLA-B* 54:01-specific CTL clone’s uniquely rearranged T cell receptor V beta chain, PCR product was detected by amplification of cDNA from pre-transplant patient peripheral blood mononuclear cells. Thus, both HLA-B* 54:01-specific antibody and CTL clone developed in the patient prior to transplantation. The present data provide the first direct evidence showing that humoral and cellular immune responses were involved in the graft rejection. Of note is the recognition of the same mismatched HLA-B* 54:01 molecule by the DSA and CTL, suggesting that humoral and cellular immune processes acted together to mediate allograft rejection. Given the difficulty in detecting HLA-specific CTLs in pre-transplant patient blood in contrast to the easiness in screening for DSAs, the presence of DSA not only means a direct deleterious effect on donor cells but it may also reflect the presence of CTLs that causes allograft rejection. Further studies are warranted to clarify whether the present observation can be duplicated in other patients with DSA. We are conducting the study to clarify whether patients with anti-HLA antibodies have CTLs that recognize the same HLA molecules before transplantation. Disclosures: No relevant conflicts of interest to declare.

In vitro and in vivo evaluations of human papillomavirus type 16 (HPV16)-derived peptide-loaded dendritic cells (DCs) with a CpG oligodeoxynucleotide (CpG-ODN) adjuvant as tumor vaccines for immunotherapy of cervical cancer

To evaluate the immunotherapeutic potentials for human dendritic cells (DCs) loaded with different HPV16-associated antigens, including HPV16E7 (E) protein, HPV16E7 polypeptide (P), as well as CpG-oligodeoxynucleotide (ODN) 2006 as a promising immune adjuvant for vaccination against cervical carcinoma. DCs derived from human peripheral blood and cord blood were isolated and loaded with HPV-derived protein or peptides, in combination with CpG-ODN2006 as a potential adjuvant. The IL-12 level, the allogeneic T cell-stimulatory capacity and the cytotoxicity of cytotoxic T lymphocytes (CTLs) were evaluated in vitro. Furthermore, an immune reconstitution model of human cervical carcinoma in SCID mice was used to assess the anti-tumor effects in vivo. The tumor sizes, the expression of IgG and IFN-γ, and the presence of the human CD3(+), CD4(+) and CD8(+) T cells were measured in the mice inoculated with different DCs. The antigen-loaded DCs displayed obvious anti-tumor activities in vitro and in vivo, and showed no toxicity to normal cells. The level of IL-12, an important cytokine for immune response, was up-regulated in all mice inoculated with antigen-loaded DCs. Stimulation and activity of CTLs were increased after treatment with antigen-loaded DCs. Significantly, DCs loaded with HPV16E7 polypeptide (P) showed the most distinguished immunotherapeutic activities, and such effect was further enhanced when HPV16E7 polypeptide (P) was used in combination with CpG-ODN2006. Interestingly, the same results were obtained in vivo: the tumor size was decreased, and IgG and IFN-γ levels were increased after the SCID mice were inoculated with the loaded DCs. HPV16E7 polypeptide combined with the immune adjuvant CpG-ODN2006 could be a suitable HPV16-associated tumor antigen. The research provides a new strategy for generating DCs vaccines for immunotherapy of cervical cancer.

Long term dynamics in a mathematical model of HIV-1 infection with delay in different variants of the basic drug therapy model

Infection with HIV-1, degrading the human immune system and recent advances of drug therapy to arrest HIV-1 infection, has generated considerable research interest in the area. Bonhoeffer et al. (1997) [1], introduced a population model representing long term dynamics of HIV infection in response to available drug therapies. We consider a similar type of approximate model incorporating time delay in the process of infection on the healthy T cells which, in turn, implies inclusion of a similar delay in the process of viral replication. The model is studied both analytically and numerically. We also include a similar delay in the killing rate of infected CD4+ T cells by Cytotoxic T-Lymphocyte (CTL) and in the stimulation of CTL and analyse two resulting models numerically.The models with no time delay present have two equilibria: one where there is no infection and a non-trivial equilibrium where the infection can persist. If there is no time delay then the non-trivial equilibrium is locally asymptotically stable. Both our analytical results (for the first model) and our numerical results (for all three models) indicate that introduction of a time delay can destabilize the non-trivial equilibrium. The numerical results indicate that such destabilization occurs at realistic time delays and that there is a threshold time delay beneath which the equilibrium with infection present is locally asymptotically stable and above which this equilibrium is unstable and exhibits oscillatory solutions of increasing amplitude.

AAV2/IL-12 gene delivery into dendritic cells (DC) enhances CTL stimulation above other IL-12 applications

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) holds significant promise in treating cancer and Th1 response cytokines are critical for their stimulation. Recently we reported that interleukin 7-(IL-7) and interferongamma-(IFNγ) autocrine/T cell gene delivery resulted in superior ex vivo CTL stimulation over paracrine/DC delivery. IL-12 is yet another important Th1 cytokine which affects both DC and T cells. Here, using adeno-associated virus Type 2 (AAV2) gene delivery, IL-12-paracrine/DC gene delivery gave significantly superior stimulation of carcinoembryonic antigen (CEA)-specific CTL killing over that induced by autocrine gene delivery (or exogenous IL-12 addition). This is surprising as both AAV2/IL-12-treated T cells and DC secreted approximately the same level of IL-12. Paracrine IL-12 gene delivery also resulted in highest IL-12/IL-10 secretion ratio by DC and highest CD40, CD80, CD83 and CD86 expression. Moreover, AAV2/IL-12-DC stimulated the highest T-cell IFNγ production, highest T cell proliferation, highest CD69+/CD8+ levels, and lowest level of CD25+/CD4+ Treg. These data strongly suggest that the primary activity of IL-12 during CTL generation is upon the DC. These data are also consistent with there being novel activity for IL-12 within the DC itself, not involving its surface receptor; an “intracrine” activity. Given the plethora of IL-12 studies, these data also suggest that this gene delivery comparison approach could be useful for uncovering new cytokine activities and mechanism(s) of action gone unrecognized by conventional immunologic assays. Finally, these data further suggest AAV2/IL-12 intracrine gene delivery into DC may have utility in immunotherapy protocols involving antigen-specific CTL.

Human Dendritic Cells Engineered to Secrete Interleukin-18 Activate MAGE-A3-Specific Cytotoxic T Lymphocytes in vitro

Adoptive cell transfer (ACT) involves the administration of tumor specific cytotoxic T lymphocytes (CTLs) into a patient to kill cancer cells. Although a promising cancer therapy, limitations on the generation of activated CTLs have restricted ATC’s clinical application. Interleukin-18 (IL-18) is an interferon-γ (IFN-γ) inducing factor that plays an important functional role in regulating CTLs. Here, we attempt to use dendritic cells (DCs) modified with a recombinant adenovirus encoding IL-18 (rAd/IL-18) to improve the generation of activated tumor-specific CTLs. These engineered DCs secrete IL-18, increase the expression of co-stimulatory molecules, and enhance the cytotoxic efficacy of melanoma antigen 3 (MAGE-A3)-specific CTLs in vitro. We show that stimulation of CTLs with rAd/IL-18-loaded DCs increases the specific lysis of MAGE-A3-expressing human breast cancer MCF-7 cells, and at the same time increases the production of activated MAGE-A3-specific CTLs. Our results indicate that transducing DCs with rAd/IL-18 increases both the maturation of DCs and the activation level of MAGE-A3-specific CTLs, greatly enhancing the cytotoxic efficacy of CTLs towards tumor cells.

Major Histocompatibility Complex-Dependent Cytotoxic T Lymphocyte Repertoire and Functional Avidity Contribute to Strain-Specific Disease Susceptibility after Murine Respiratory Syncytial Virus Infection

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

Abstract LB-207: IFN-Γ directly regulates ifi202B expression to maintain CTL proliferation potential and tumor rejection efficacy

Abstract A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer cancer immunotherapy is to propagate tumor-specific CTLs in vitro using anti-CD3 mAb prior to transfer. One of the major impediments of CTL adoptive cancer immunotherapy is lack of CTL persistence in the tumor-bearing host after adoptive transfer. Therefore, maintaining CTL proliferation and persistence in the tumor microenvironment is of great significance in cancer immunotherapy. In this study, we observed that stimulation of tumor-specific CTLs with anti-CD3 mAb results in loss of proliferative potential and poor tumor-rejection efficacy in vivo, whereas, stimulation with tumor-specific antigen maintains CTL proliferative potential and persistence, leading to potent tumor rejection in an experimental lung metastasis mouse model. Analysis of genome-wide gene expression profile revealed that ifi202B, a gene with known function in regulating T cell proliferation and apoptosis, was dramatically up-regulated in antigen-stimulated but not in anti-CD3 mAb-stimulated CTLs. Furthermore, although both antigen- and anti-CD3 mAb-stimulated CTLs express similar level of IFN-Γ receptor, antigen stimulation led to rapid activation of STAT1, a key mediator of the IFN-Γ signaling pathway, whereas anti-CD3 mAb stimulation failed to activate STAT1. We further demonstrated that activated STAT1 binds to the promoter of ifi202b gene in T cells. Taken together, these observations suggest that IFN-Γ produced by the activated CTLs and the IFN-Γ signaling pathway plays a critical role in maintaining CTL proliferation and persistence in the tumor microenvironment through regulating ifi202B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-207.

Autocrine, Not Paracrine, Interferon-Gamma Gene Delivery Enhances Ex Vivo Antigen-Specific Cytotoxic T Lymphocyte Stimulation and Killing

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon γ (IFN-γ), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-γ is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-γ gene delivery into DC or T cells, and versus the addition of exogenous IFN-γ, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-γ delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-γ(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-γ transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-γ autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers.

Multivirus-Specific T Cell Immunotherapy to Prevent or Treat Infections of Stem Cell Transplant Recipients

Viral infections cause morbidity and mortality in allogeneic HSCT recipients. To treat the underlying problem, namely lack of antigen-specific T cells, we and others have successfully generated and infused adoptive T-cell lines specific for EBV, CMV and Adenovirus. We have shown that as few as 2 × 105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs post-infusion and appeared to protect the recipients against all three viruses. Despite the encouraging clinical results, broader implementation of the approach is limited by (i) the usage of infectious virus material (EBV/Adv) and (ii) the prolonged culture (3 months for EBV-LCL production and CTL stimulation).

RAAV/Her-2/neu Loading of Dendritic Cells for a Potent Cellular-Mediated MHC Class I Restricted Immune Response Against Ovarian Cancer

Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates, in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. We used the rAAV system to induce specific CTLs against tumor antigens for the development of ovarian cancer (OC) gene therapy. As an extension of the versatility of the rAAV system, we incorporated a self-antigen, Her-2/neu, which is expressed in many cancers, including breast and ovarian. We analyzed two different vectors containing a short (157-612) and long domain (1-1197). Our rAAV vector induced strong stimulation of CTLs directed against the self tumor antigen, Her-2/neu. We then investigated the efficiency of the CTLs in killing Her-2/neu-targeted cells. A significant MHC class I-restricted, anti-Her-2/neu-specific CTL killing was demonstrated against Her-2/neu-positive OC cells after one in vitro stimulation. In summary, single peripheral blood mononuclear cell (PBMC) stimulation with rAAV/157-612- or rAAV/1-1197-pulsed DCs induces strong antigen-specific CTL generation. The CTLs were capable of lysing low doses of peptides pulsed into target cells or OC Her-2/neu(+) tumors. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against OC antigens.

Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

BackgroundRecent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.MethodsWe used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.ResultsA significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.ConclusionIn summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.

Fusion of B Lymphoblastoid and Tumor Cells Expressing Different Antibiotic Resistance Genes Facilitates Selection of Stable Hybridomas

Abstract The development of tumor vaccines or generation of tumor-specific cytotoxic T lymphocytes (CTL) is limited by the fact that many tumor cells downregulate the expression of major histocompatibility complex (MHC) Class I and II molecules, as well as key co-stimulatory molecules such as CD80 and CD86. An immune response to a vaccine or in vitro stimulation of tumor-specific CTL requires antigen-presenting cells conveying tumor antigens in the context of a host's MHC antigens. We have used a retroviral vector (murine stem cell virus) encoding neomycin resistance to transduce three pediatric tumor cell lines (two neuroblastoma, one neuroepithelial tumor). An EBV transformed B lymphoblastoid cell line (BLCL) was transduced with a separate vector encoding puromycin resistance and green fluorescent protein, individual tumor lines were fused with the BLCL, and the resulting hybridomas were selected using both antibiotics. The resulting hybridoma cells expressed the neural antigen GD2 as well as MHC Class I, Class II, CD 80, and CD86. A similar strategy could be used to produce stable hybridomas for either vaccination or for CTL expansion.