CD8 OT-I Research Articles

Sepsis Impairs IFN-γ Production in CD8 T Cells through Changes in Local Chromatin Landscape.

Sepsis is a complex condition of inflammatory and immune dysregulation, triggered by severe infection. In survivors, chronic inflammation and immune dysregulation linger, facilitating the emergence of infections. CD8 dysfunction contributes to immunosuppression in sepsis survivors. We devised an animal model that enabled us to identify and analyze CD8-intrinsic defects induced by sepsis. We adoptively transferred CD45.1 CD8 OT-I T cells into CD45.2 congenic mice and subjected them to cecal ligature and puncture, to induce abdominal sepsis. One month later, we isolated the transferred CD8 cells. Surface marker expression confirmed they had not been activated through the TCR. CD8 OT-I T cells isolated from septic (or sham-operated) mice were transferred to second recipients, which were challenged with OVA-expressing Listeria monocytogenes. We compared effector capacities between OT-I cells exposed to sepsis and control cells. Naive mice that received OT-I cells exposed to sepsis had higher bacterial burden and a shorter survival when challenged with OVA-expressing L. monocytogenes. OT-I cells isolated from septic mice produced less IFN-γ but had conserved activation, expansion potential, and cytotoxic function. We observed lower transcript levels of IFN-γ and of the long noncoding RNA Ifng-as1, a local regulator of the epigenetic landscape, in cells exposed to sepsis. Accordingly, local abundance of a histone modification characteristic of active promoter regions was reduced in sepsis-exposed CD8 T cells. Our results identify a mechanism through which inflammation in the context of sepsis affects CD8 T cell function intrinsically.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and Tcell activation against a vesicularstomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) invitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I Tcells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 Tcell response invivo without altering the anti-viral CD8 Tcell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

Vesicular stomatitis virus expressing destabilized tumor associated antigens with enhanced MHC class I presentation to improve anti-tumor T cell responses

Abstract We have previously shown that vesicular stomatitis virus (VSV) vectors encoding truncated tumor antigens, NRAS, Cytochrome C1, and TYRP1, prime superior systemic anti-tumor T cell responses and provide enhanced therapeutic benefit compared to vectors expressing full-length forms of the protein. Mapping of the roughly 20 amino C-terminal truncated regions onto structural domains of each protein suggested the truncations disrupted the stability and localization of each protein. Indeed, immunofluorescent staining and radiolabeled pulse chase found that truncated TYRP1 (ΔTYRP1) had an altered localization pattern and a significantly reduced half-life compared to full-length TYRP1. Thus, we hypothesized that expression of destabilized antigens from VSV would provide superior therapy compared to expression of wild-type antigens. To test this, we introduced point-mutations into structurally important domains of the model antigen ovalbumin (OVA). We identified one mutant which was significantly destabilized (OVAmut), which resulted in enhanced MHC Class I presentation of the immunogenic SIINFEKL epitope on transfected cell in vitro. Expression of OVAmut resulted in better recognition by cognate CD8 OTI T cells, enhanced T cell activation, proliferation, and effector cytokine production. Finally, a VSV vector expressing OVmut was better able to prime OTI T cells in vitro as well as in vivo. Ongoing studies aim to identify the changes in the quantity and repertoire of immunogenic epitopes presented from cells infected with VSV vectors expressing destabilized antigens. These studies provide insight into the capacity of the VSV platform to successfully encode unstable antigens with the goal improve immunogenic responses.

CD8 T cells primed in the gut-associated lymphoid tissue induce immune-mediated cholangitis in mice

The pathogenesis of primary sclerosing cholangitis (PSC) remains poorly understood. Since PSC predominantly occurs in patients with inflammatory bowel disease, autoimmunity triggered by activated T cells migrating from the gut to the liver is a possible mechanism. We hypothesized that T cells primed in the gut-associated lymphoid tissue (GALT) by a specific antigen migrate to the liver and cause cholangitis when they recognize the same antigen on cholangiocytes. We induced ovalbumin-dependent colitis in mice that express ovalbumin in biliary epithelia (ASBT-OVA mice) and crossed ASBT-OVA mice with mice that express ovalbumin in enterocytes (iFABP-OVA mice). We analyzed T-cell activation in the GALT and crossreactivity to the same antigen in the liver as well as the effects of colitis per se on antigen-presentation and T-cell activation in the liver. Intrarectal application of ovalbumin followed by transfer of CD8 OT-I T cells led to antigen-dependent colitis. CD8 T cells primed in the GALT acquired effector function and the capability to migrate to the liver, where they caused cholangitis in a strictly antigen-dependent manner. Likewise, cholangitis developed in mice expressing ovalbumin simultaneously in biliary epithelia and enterocytes after transfer of OT-I T cells. Dextran sodium sulfate colitis led to increased levels of inflammatory cytokines in the portal venous blood, induced activation of resident liver dendritic cells, and promoted the induction of T-cell-dependent cholangitis. Our data strengthen the notion that immune-mediated cholangitis is caused by T cells primed in the GALT and provide the first link between colitis and cholangitis in an antigen-dependent mouse model.

Abstract A32: Combining early heat shock protein vaccination with directed IL-2 leads to effective and persistent antitumor immunity in recipients of experimental autologous hematopoietic cell transplantation.

Abstract Tumor relapse remains the major cause of morbidity and mortality in patients with hematologic cancers after aggressive chemo-radiotherapy and autologous hematopoietic cell transplant (HCT) rescue. Therefore, vaccination strategies can be employed early post-HCT during ‘reboot’ of the immune system to mold anti-tumor immunity by taking advantage of both minimal residual disease and lymphopenia. The pre-clinical studies here developed a new strategy uniquely suitable to this environment. We reasoned that by introducing antigen as a heat shock protein (hsp) vaccine, tumor specific CD8 T cells would be rapidly elicited. Subsequently, administration of IL-2 ‘directed’ to CD8 T cells would augment vaccine induced expansion and survival of lymphoma bearing HCT recipients. Recipient B6 mice were therefore conditioned with myeloablative total body irradiation prior to syngeneic HCT. To more precisely model clinical transplants, T cells were obtained from syngeneic donors bearing progressively growing lymphoma (EG7). Transplanted mice were inoculated with a low number of EG7 cells to simulate tumor relapse post-HCT. A multiple vaccination protocol was employed utilizing irradiated EG7 cells transfected to secrete the hsp fusion protein gp96-Ig (EG7-gp96-Ig). To ‘direct’ IL-2 to CD8 T cells, we infused complexes of IL-2 pre-bound to αIL-2 mAb clone S4B6 (IL-2S4B6). Transplanted animals not vaccinated or vaccinated with parental EG7 cells exhibited an MST of ~1 month with 100% lethality. In contrast, vaccination with EG7-gp96-Ig cells significantly extended MST and increased overall survival to 20% (>100 d post-HCT). Enhanced MST was dependent on transplant of donor T cells since vaccination failed to prolong survival in recipients of T cell depleted marrow grafts. Strikingly, combination therapy with EG7-gp96-Ig and IL-2S4B6 complex resulted in a dramatic increase of overall survival (60%) and MST. Treatment with either: a) unbound ‘free’ IL-2 in combination with vaccine, or b) IL-2S4B6 complex monotherapy (no vaccine), did not induce a survival advantage vs. vaccine therapy alone. Experiments are ongoing to determine which components of the donor graft are required for prolonged survival. Surviving mice (>100 d post-HCT) were re-challenged with a lethal number of EG7 cells. Most HCT recipients (n = 16) vaccinated with EG7-gp96-Ig cells alone or in combination with IL-2S4B6 complex rejected the tumor (87.5%), illustrating the presence of long term anti-tumor memory. To monitor tumor specific T cells, CD8 OT-I were transferred to some donor mice preceding tumor inoculation. This resulted in ~1000 OT-I present in the 2x106 donor T cell inoculum used for HCT. Importantly, no effect on overall survival of HCT recipients was observed regardless of the presence or absence of the transplanted OT-I. Treatment with hsp vaccine and ‘directed’ IL-2 resulted in ~36,000x expansion of OT-I within 2 wk of HCT, compared to ~7000x after vaccination alone. OT-I at the vaccine site produced IFN-γ and TNF-α after ex vivo peptide stimulation. Notably, the combination strategy elicited large increases in both donor CD8 T and NK cells, populations essential for pathogen immunity and vaccine efficacy. Experiments are ongoing to determine if a 2 cycle protocol, administered in part during de novo T cell genesis (>3 wk post-HCT), can elicit additional immunity from these newly derived T cells. Vaccination approaches employing tumor cells secreting gp96-Ig are presently in clinical trials for lung cancer utilizing an ‘off the shelf’ modified allogeneic lung cancer cell line. Interestingly, others have engineered a human IL-2 “superkine”, resulting in a binding pattern similar to the cytokine/antibody complex used in the present studies. Thus, the combinatorial regimen developed here represents a promising strategy with translational applications for patients with hematologic cancers. Citation Format: Robert G. Newman, Eckhard R. Podack, Robert B. Levy. Combining early heat shock protein vaccination with directed IL-2 leads to effective and persistent antitumor immunity in recipients of experimental autologous hematopoietic cell transplantation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A32.

CD8 T Cell Expansion Early Post-Autologous HSCT Following Vaccination with Lymphoma Cells Secreting gp96-Ig and Augmentation by IL-2 Antibody Cytokine Complex

AbstractAbstract 4286In patients with hematologic cancers, such as lymphoma, aggressive radiation and chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) can achieve a minimal residual disease state. Nonetheless, relapse remains the major cause of morbidity and mortality in such patients, and there remains a critical need for more effective anti-tumor therapy. Vaccination with tumor cells secreting the heat shock fusion protein gp96-Ig can induce robust CTL expansion in non-transplanted mice, however its efficacy in HSCT recipients where the opportunity to manipulate the immune response during hematolymphoid reconstitution has not been studied. We examined strategies to expand antigen specific CD8 T cells using lymphoma cells secreting gp96-Ig heat shock fusion protein in the early post auto-HSCT setting. B6 mice (CD45.2+) received ablative conditioning (9.5 Gy TBI) followed by the infusion of 5×106 congenic (CD45.1+) T cell depleted BMC. To investigate vaccine timing, E.G7-OVA lymphoma cells secreting gp96-Ig (E.G7-gp96-Ig: 107/120 Gy) were introduced either 2 or 7 days following auto-HSCT. Two days prior to vaccination, recipients received 106 CD8 OT-I T cells. Following single intraperitoneal E.G7-gp96-Ig vaccination, OT-I expansion occurred at the site of vaccination, reaching maximal levels 5 days post-vaccination. Expansion was clearly superior following vaccination on day 7. Multiple vaccinations (day 7 and 13) improved this response including enhancement of OT-I accumulation in the spleen and LN. Greater than 80% of the OT-I CD8 T cells displayed an antigen-experienced effector memory phenotype (CD44hiCD62Llo) after vaccination with E.G7-gp96-Ig. Notably, little OT-I expansion occurred following vaccination with parental E.G7-OVA (Fig 1), and the OT-I present displayed a central memory phenotype (CD44hiCD62Lhi). Since gp96 chaperoned peptides are cross-presented by APC, we determined the source of B7.1/2 costimulation with regard to donor and recipient APC. Interestingly, following day 7 vaccination CD8 OT-I expansion was markedly diminished when donor BMC were transplanted from B7.1/2 KO mice into WT or B7.1/2 KO recipients (Fig 1). In contrast, following day 2 vaccination, recipient B7.1/2+ APC clearly contributed to OT-I expansion. These observations are consistent with the transition to predominant donor APC presence early following auto-HSCT. To attempt to enhance vaccine induced CD8 expansion, immune complexes comprised of IL-2 and anti-IL-2 mAb clone S4B6 (IL-2/S4B6) were administered following day 7 vaccination. This specific IL-2 immune complex has been shown to interact with CD8 T cells expressing the intermediate affinity IL-2 receptor (IL-2Rβγ), which is expressed on memory CD8 T (concomitantly expressing CD44) and NK cells. Combination treatment with E.G7-gp96-Ig and IL-2/S4B6 immune complex resulted in 4x greater expansion of OT-I T cell numbers at the site of vaccination and in the spleen versus E.G7-gp96-Ig vaccination alone. Thus, 1 week following vaccination, >6×106 OT-I were identified in the compartments analyzed. Interestingly, residual host CD8 T cells were also expanded following complex administration. In summary, we hypothesize that gp96-Ig vaccination post-HSCT results in cross presentation of tumor peptides to CD8 T cells by mature or newly derived B7.1/2 expressing APC of donor origin. Addition of IL-2 via specific mAb/cytokine immune complex provides a strategy to augment gp96-Ig induced CD8 expansion. Studies are underway to address the efficacy of this approach in pre-clinical lymphoma models involving T cell replete auto-HSCT. [Display omitted] Disclosures:Podack:Heat Biologics, Inc.: Consultancy, Equity Ownership.