OT-I Cells Research Articles

IL-1 enhances expansion, effector function, tissue localization, and memory response of antigen-specific CD8 T cells

Here, we show that interleukin-1 (IL-1) enhances antigen-driven CD8 T cell responses. When administered to recipients of OT-I T cell receptor transgenic CD8 T cells specific for an ovalbumin (OVA) peptide, IL-1 results in an increase in the numbers of wild-type but not IL1R1−/− OT-I cells, particularly in spleen, liver, and lung, upon immunization with OVA and lipopolysaccharide. IL-1 administration also results in an enhancement in the frequency of antigen-specific cells that are granzyme B+, have cytotoxic activity, and/ or produce interferon γ (IFN-γ). Cells primed in the presence of IL-1 display enhanced expression of granzyme B and increased capacity to produce IFN-γ when rechallenged 2 mo after priming. In three in vivo models, IL-1 enhances the protective value of weak immunogens. Thus, IL-1 has a marked enhancing effect on antigen-specific CD8 T cell expansion, differentiation, migration to the periphery, and memory.

Persistent Antigen and Germinal Center B Cells Sustain T Follicular Helper Cell Responses and Phenotype

T follicular helper (Tfh) cells provide help to B cells and are crucial for establishment of germinal center (GC) reactions, including production of high-affinity antibodies and generation of memory B cells and long-lived plasma cells. Here we report that the magnitude of the Tfh cell response was dictated by the amount of antigen and directly correlated with the magnitude of the GC B cell response. In addition, maintenance of the Tfh cell phenotype required sustained antigenic stimulation by GC B cells. In lymphopenic conditions, a strong and prolonged Tfh cell response led to bystander B cell activation, hypergammaglobulinemia, and production of poly- and self-reactive antibodies. These data demonstrate that antigen dose determines the size and duration of the Tfh cell response and GC reaction, highlight the transient nature of the Tfh cell phenotype, and suggest a link between overstimulation of Tfh cells and the development of dysregulated humoral immune responses.

A catalytically inactive mutant of the deubiquitylase YOD-1 enhances antigen cross-presentation

AbstractAntigen presenting cells (APCs) that express a catalytically inactive version of the deubiquitylase YOD1 (YOD1-C160S) present exogenous antigens more efficiently to CD8+ T cells, both in vitro and in vivo. Compared with controls, immunization of YOD1-C160S mice led to greater expansion of specific CD8+ T cells and showed improved control of infection with a recombinant γ-herpes virus, MHV-68, engineered to express SIINFEKL peptide, the ligand for the ovalbumin-specific TCR transgenic OT-I cells. Enhanced expansion of specific CD8+ T cells was likewise observed on infection of YOD1-C160S mice with a recombinant influenza A virus expressing SIINFEKL. YOD1-C160S APCs retained antigen longer than did control APCs. Enhanced cross-presentation by YOD1-C160S APCs was transporter associated with antigen processing (TAP1)–independent but sensitive to inclusion of inhibitors of acidification and of the proteasome. The activity of deubiquitylating enzymes may thus help control antigen-specific CD8+ T-cell responses during immunization.

Abstract B68: Aptamer-targeted siRNA-mediated inhibition of mTORC1 in antigen-primed CD8+ cytotoxic T lymphocytes enhances immunological memory and antitumor immunity.

Abstract Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Multiple extrinsic pathways control the differentiation of CD8+ cytotoxic T lymphocytes (CTL) into effector or memory cells by regulating the balanced expression of intracellular mediators in the antigen-activated T cells. Notably, inhibition of mediators of effector differentiation like mTOR, GSK3β or T-bet using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR or GSK3β in mice with rapamycin or TSW119, respectively, led to the differentiation of antigen activated CD8+ T cells into long lasting memory cells that exhibited enhanced antiviral and antitumor immunity. Nevertheless, pharmacological agents often exhibit undesirable effects reflecting the broad distribution of their targets. For example, rapamycin inhibition of mTOR promotes the development of immune suppressive regulatory CD4+ T cells and GSK3β; inhibition polarizes dendritic cells (DC) to a tolerogenic state, arguably counterproductive in the setting of vaccination. In addition, development of pharmacological agents to modulate the function of intracellular targets that are not accessible to antibodies (“undruggable targets”) is highly challenging, and their availability especially for clinical use is limited. Here we describe a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi was used to downregulate intracellular mediators of effector differentiation that were targeted to CD8+ T cells by conjugation to aptamer ligands. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice by tail vein injection downregulated mTORC1, while preserving mTORC2, activity in at least 60% of adoptively transferred OVA-specific transgenic OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory response in mice as judged by the ability of the memory cells to proliferate in response to re-exposure to antigen at a latter time point. However, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. In contrast, the aptamer-raptor siRNA generated CTL exhibited normal cytotoxic effector functions and enhanced vaccine-induce protective antitumor immunity in both prophylactic and therapeutic tumor models. Reflecting the lack of cell specificity of mTOR inhibition by rapamycin, and providing a potential mechanisms underlying the effector defect of rapamycin generated CTL, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced MLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC. These studies demonstrate the feasibility and efficiency of aptamer targeted delivery of siRNAs to immune cells, and underscore the potential advantages of aptamer-targeted siRNA delivery over nontargeted administration of pharmacological agents. Citation Format: Alexey Berezhnoy, Iris Castro, Agata Levay, Thomas Malek, Eli Gilboa. Aptamer-targeted siRNA-mediated inhibition of mTORC1 in antigen-primed CD8+ cytotoxic T lymphocytes enhances immunological memory and antitumor immunity. [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 B68.

Abstract B23: DNA damage response in responder lymphocytes reduces vaccine efficacy following alkylating chemotherapy.

Abstract The effect of alkylating chemotherapy on cancer vaccines is controversial, with numerous reports of immune stimulation via Treg depletion following cyclophosphamide and temozolomide treatment. Using a pre-clinical model of a cancer vaccine in both naïve and tumor bearing animals, we observed that the direct anti-proliferative effect of drugs such as temozolomide or cyclophosphamide reduced the quantity of vaccine induced immune responses in a dose dependent manner. Amounts of temozolomide exposure in mice that duplicated human pharmacokinetic exposure and lymphopenia led to lower numbers of CD8 T cells elicited by vaccination with the model antigen ovalbumin. In vitro experiments with purified OT-I CD8 T cells implicated activation of DNA damage responses within strongly proliferating cells as the cause of this diminished immunity. ATM phosphorylation was greater in temozolomide or cyclophosphamide exposed OT-I cells stimulated with the canonical SIINFEKL peptide relative to variant peptides that trigger weaker TCR signaling. In vaccinated mice, this activation of DNA damage response in lymphocytes receiving the highest intensity TCR signals lead to lower avidity of responder cells for cognate antigen and lower effector function. These quantitative and qualitative defects of vaccine driven responses lead to inferior efficacy and loss of survival benefit in murine models. Mice bearing ovalbumin peptide expressing glioma had faster tumor growth and shorter survival following temozolomide and vaccine relative to vaccine only treated mice. Similar defects of vaccine generated responses and a loss of survival benefit relative to controls were observed in response to a neo-antigen vaccine following cyclophosphamide pre-treatment in melanoma bearing mice. The clinical relevance of this finding is demonstrated by a phase II cancer vaccine trial in metastatic melanoma patients where vaccine only patients survived a median of 4.2 years versus less than 8 months in patients treated with low dose cyclophosphamide before vaccine. Citation Format: Adam J. Litterman, David M. Zellmer, Karen L. Grinnen, Arkadiusz Z. Dudek, David A. Largaespada, John R. Ohlfest. DNA damage response in responder lymphocytes reduces vaccine efficacy following alkylating chemotherapy. [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 B23.

CD4 Cells Engineered to Express an MHC Class I Restricted TCR Can Rescue CD8 Cells Tolerized to Tumour-Associated Antigens

AbstractAbstract 952 Background:The efficacy of T cell therapies for cancer may be limited when targeting tumour-associated antigens (TAA) which are also self-antigens. Ongoing exposure to TAA on normal cells may lead to tolerance via anergy or exhaustion of antigen-specific T cells. Methods:We have designed a model of tolerance to TAA in which T cell receptor (TCR)-transduced CD8 T cells recognise pMDM2, a TAA that is also a ubiquitous self-antigen. CD8+ T cells were transduced with pMDM2-specific TCR (MDM-CD8) and transferred to sub-lethally irradiated B6 mice that express pMDM2 in the context of MHC Class I (H2-Kb). MDM-CD8 cells are detectable 4 weeks after transfer but show defective in vivo killing of target cells pulsed with MDM2 peptide. We have used this model to determine the mechanism of tolerance and to evaluate whether tolerant CD8+ T cells can be rescued by CD4 help. Results:To determine whether tolerance of MDM-CD8 cells was dependent upon recognition of cognate antigen, we transferred MDM-CD8 cells into mice of a different MHC background (BALB/c) which lack H2-Kb required for presentation of the TCR-recognised MDM2 peptide. When BALB/c MDM-CD8 cells were transferred to BALBc hosts their functions were preserved and they retained efficient antigen-specific cytolysis.To determine whether tolerance could be modified by provision of CD4+ T cell help, we co-transferred MDM-CD8 with transgenic OT-II CD4+ cells. OT-II cells were primed with dendritic cells (DCs) loaded with cognate pOVA323-339 or irrelevant peptide. When activated through their TCR, OT-II cells increased both the frequency of MDM2-specific CD8 cells and their cytotoxic functions, indicating that CD4 help can overcome CD8 tolerance to TAA.Ineffective antigen presentation to CD4 cells and lack of known MHC class II-restricted TAA are major limitations to providing CD4 help in T cell therapy for cancer. We therefore tested whether transfer of the MHC Class I-restricted MDM2 TCR into CD4 cells could provide help upon transfer to antigen-expressing hosts. Co-transfer of MDM2-TCR-transduced CD4 cells with CD8 cells improved antigen-specific killing of target cells when compared to single transfer of either TCR-transduced CD8 or CD4 cells. Conclusion:CD4 cells rendered capable of responding to an MHC class I restricted TAA by TCR transfer can rescue tolerance developing in a CD8 population with the same specificity. This is potentially a novel way to circumvent defective immune responses arising in adoptively transferred effector cells due to prolonged exposure to cognate antigen on normal host cells. Disclosures:Stauss:Cell Medica: Scientific Advisor Other.

Development of Memory CD8+ T Cells and Their Recall Responses during Blood-Stage Infection with Plasmodium berghei ANKA

Conditions required for establishing protective immune memory vary depending on the infecting microbe. Although the memory immune response against malaria infection is generally thought to be relatively slow to develop and can be lost rapidly, experimental evidence is insufficient. In this report, we investigated the generation, maintenance, and recall responses of Ag-specific memory CD8(+) T cells using Plasmodium berghei ANKA expressing OVA (PbA-OVA) as a model system. Mice were transferred with OVA-specific CD8(+) T (OT-I) cells and infected with PbA-OVA or control Listeria monocytogenes expressing OVA (LM-OVA). Central memory type OT-I cells were maintained for >2 mo postinfection and recovery from PbA-OVA. Memory OT-I cells produced IFN-γ as well as TNF-α upon activation and were protective against challenge with a tumor expressing OVA, indicating that functional memory CD8(+) T cells can be generated and maintained postinfection with P. berghei ANKA. Cotransfer of memory OT-I cells with naive OT-I cells to mice followed by infection with PbA-OVA or LM-OVA revealed that clonal expansion of memory OT-I cells was limited during PbA-OVA infection compared with expansion of naive OT-I cells, whereas it was more rapid during LM-OVA infection. The expression of inhibitory receptors programmed cell death-1 and LAG-3 was higher in memory-derived OT-I cells than naive-derived OT-I cells during infection with PbA-OVA. These results suggest that memory CD8(+) T cells can be established postinfection with P. berghei ANKA, but their recall responses during reinfection are more profoundly inhibited than responses of naive CD8(+) T cells.

α-1 Antitrypsin Promotes Semimature, IL-10–Producing and Readily Migrating Tolerogenic Dendritic Cells

Tolerogenic IL-10-positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7-dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. α-1 antitrypsin (AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4(+) T cells, and AAT in vitro and in vivo. IL-1β/IFN-γ-mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT (0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and maintained inducible CCR7. Upon exposure of CD4(+) T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3(+) Treg cells were obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60 mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection, whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group. Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT.

Unlike CD4+ T‐cell help, CD28 costimulation is necessary for effective primary CD8+ T‐cell influenza‐specific immunity

The importance of costimulation on CD4(+) T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4(+) T-cell help. The role of costimulation under such "helpless" circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44(+) BrdU(+) influenza-specific CD8(+) T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin-specific transgenic CD8(+) T cells (OT-I)-I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT-I cell division. As shown previously, neither viral clearance nor the generation of influenza-specific CD8(+) T cells was affected by the absence of CD4(+) T cells alone. In contrast, both were markedly impaired by CD28 blockade of "helpless" CD8(+) T cells. We suggest that direct CD28 costimulation of CD8(+) T cells is more critical in their priming during primary influenza infection than previously appreciated.

Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by Purging the Peripheral Repertoire of Autoreactive CD8+ T Cells

Myelin-specific CD8(+) Tcells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) Tcells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) Tcells failed to induce disease and were cleared from the immune system after antigen encounter inthe centralnervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) Tcells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) Tcells to cause demyelinating disease. Thus, in response to infection, CD8(+) Tcells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

Effective prevention and treatment of CD8 T cell-mediated graft-versus-host-like disease (GvHD) using a JAK inhibitor. (51.3)

Abstract A Janus kinase (JAK) inhibitor, tofacitinib (tofa), inhibits JAK 1, 2 and 3 of some cytokine receptor pathways, affects CD4+ T cell respnses, and is being tested in more than 20 clinical trials in autoimmune diseases.To determine if tofa affects CD8+ T cells, we analyzed its effect on a CD8+ T-cell-dependent murine model of GvHD (OT-I T cell transfer into Tg mice expressing ovalbumin (OVA) in their skin and mucosae). Tofa at 50 mg/kg/day (50) p.o. completely prevented GvHD development in our model vs. vehicle-treated mice with final weights: -0.6%±5.6 and -15.4%±4.1 (p<0.01; 2-way ANOVA) and clinical scores: 0 and 7.3±0.52 (p<0.01; U test) 13 days post OT-I cell transfer. Mice treated with tofa 50 starting at day 5 were completely rescued, with no clinical signs of GVHD. FACS analysis demonstrated reduced expression of OVA-specific TCR and reduced activation of OT-I cells in skin-draining lymph nodes from mice transferred with GFP/OT-I cells and treated with tofa 50 vs. vehicle alone. Tofa inhibited proliferation of OT-I cells stimulated by SIINFEKL (OVA peptide) in vitro. qRT-PCR showed increased chemokine mRNA (CXCL9; 39-fold and CXCL10; 22-fold) in the epidermis of vehicle-treated mice vs. tofa 50-treated mice 4 days post OT-I cell transfer. The effect of tofa could be explained by its inhibition of JAK signaling in IL-2 and IL-15 receptor-pathways of OT-I cells and in IFN-γ receptor-pathways of keratinocytes and tofa may thereby be effective in autoimmune diseases.

Differential requirement for CCR7 expression in primary and secondary CD8 T cell responses to infection (69.9)

Abstract Since naïve and memory T cells localize in both red and white pulp of the spleen, we wished to test the relative contribution of priming in each location. To do so, we employed TCR transgenic CD8 T cells (OT-I) that either lacked CCR7 or constitutively expressed CCR7 (CD2-CCR7). After Listeria infection, CCR7-/- CD8 T cells did not enter the T cell zone (PALS) while CD2-CCR7 OT-I cells were confined nearly exclusively to the PALS. The response of CCR7-/-CD8 T cells was blunted compared to WT and CD2-CCR7 OT-I cells and was characterized by skewing toward KLRG1highCD127low short-lived effector cells (SLEC) and away from KLRG1lowCD127high memory precursor effector cells (MPEC). Transfer studies showed that CCR7-/- OT-I cells competed with endogenous ova-specific CD8 T cells, suggesting that some priming occurred in the red pulp in normal conditions. Interestingly, CCR7-/-, CD2-CCR7 and WT OT-I memory cells responded equally well to challenge infection indicating a low stringency for localization for memory CD8 T cell activation. These results suggested that antigen presentation in the red pulp promoted SLEC generation while white pulp priming promoted MPEC development, perhaps due to differences in the inflammatory milieu. Along with our demonstration that naïve and memory CD8 T cell activation exhibited distinct anatomical requirements, our findings implied that local environmental cues regulated the development of primary and secondary CD8 T cell responses to infection.

Enhanced memory development in mice by aptamer targeted mTORC1 inhibition in CD8+ T cells (110.3)

Abstract Recent studies have highlighted the importance of immunological memory in mediating protective immunity in chronic infectious diseases and cancer. Inhibition of intracellular mediators of T cell differentiation with pharmacological agents was shown to promote memory differentiation in mice. However, given their often pleiotropic effects, non-targeted administration of pharmacological agents could compromise their immune promoting properties. We have shown that a CD8+ T cell targeted 4-1BB aptamer -raptor siRNA conjugate downregulated mTORC1, while preserving mTORC2, activity in 60% of adoptively transferred OVA-primed OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced proliferative recall response. However, the rapamycin-generated memory cells were defective in their in vivo cytotoxic effector functions which correlated with reduced perforin expression. Likewise, rapamycin or aptamer-siRNA conjugate treatment enhanced the in vivo differentiation of gp100-primed Pmel-1 cells into memory cells. Consistent with a cytotoxic defect in the rapamycin-generated memory cells, the rapamycin-treated mice were not able to inhibit a challenge with gp100-expressing B16 melanoma tumor cells. These observations support the premise that in vivo cell-targeted inhibition of mTOR, and conceivably other mediators of T cell differentiation, will circumvent undesirable effects of non-targeted inhibition by pharmacological agents.

Soluble Peptide Treatment Reverses CD8 T-Cell-Induced Disease in a Mouse Model of Spontaneous Tissue-Selective Autoimmunity

Transgenic (Tg) mouse models of autoimmunity have been used to express model antigens that can be recognized by T cells or by autoantibodies. To identify mechanisms of CD8-mediated tissue-specific autoimmune reactions and to identify potential treatments, we generated a double-transgenic (DTg) murine model of autoimmunity by crossing keratin-14 (K14)-soluble chicken ovalbumin (sOVA) mice, which express sOVA predominantly in external ear skin, with OT-I mice whose CD8 T cells express Vα2/Vβ5 regions of the TCR and are specific for SIINFEKL peptide (chicken ovalbumin (OVA) peptide 257-264) in association with class I major histocompatibility complex. The K14-sOVA/OT-I DTg mice develop a destructive process selectively targeting the external ear pinnae in the first 6 days of life. The ear bud area develops an intense inflammatory infiltrate of OT-I cells. Administration of the SIINFEKL peptide intravenously to pregnant F1 (filial 1, first filial generation of animal offspring from cross-mating two parental types) mice and subsequently intraperitoneally to newborn pups resulted in normal external ear development. Treatment with this self-peptide markedly reduced OT-I cell numbers, as well as downregulated the CD8 co-receptor. This model can be useful in studying localized, tissue-specific, immune-mediated skin disease, and provide information about potential therapies for autoimmune diseases in which specific molecular targets are known.

Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs) is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm) are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

Development of a Novel Method for in Vitro Analysis of CD8 Thymocyte Selection and Maturation,

Abstract 3235T cell development relies on the interaction between the T-cell receptor (TCR) on thymocytes and the self-major histocompatibility complex (MHC) expressed on thymic epithelial cells in the thymus. This process, called positive selection, rescues developing thymocytes from cell death while leading to their differentiation into mature T cells. Since it is believed that the proper development of CD8 T cells requires an intact thymus, several groups studied their development using fetal or reaggregation thymus organ cultures in vitro. Unfortunately, these models were shown to be cumbersome requiring a complicated set-up while generating limited cellular yield. Thus, we sought of developing a novel in vitro system using bone marrow-derived stromal cells to support CD8 T cell development and maturation in vitro. We selected the OTI system as a working model due to the availability of previously identified positively selecting peptides. Non-selected T-cell-committed double-positive (DP) OTI thymocytes (CD4+CD8+CD69−) were first fractionated based on the surface expression intensity of both TCR and CD5. These 3 subsets designated as TCRloCD5lo (DP1), TCRintCD5hi (DP2), and TCRhiCD5int (DP3) express different levels of ZAP70. Following fractionation, the DP subsets were co-cultured with bone marrow-derived stromal cells presenting OTI-selecting peptides. In the absence of cytokines, no CD8+ OTI cell development occurred in vitro. When repeated in the presence of γc-cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) only rIL4 and rIL7 were able to induce CD8 T cell development. Supplementing the co-culture system with rIL4 led to the generation of 50–60% single-positive (SP) CD8 T cells only from the DP3 fraction whereas rIL7 induced the development of a minor fraction of CD8 T cells from DP2s (3–4%) and a major population from DP3 (50–76%). Furthermore, we found that rIL4 treatment triggers the development of 2 distinct populations of SP OTI cells (based on their CD8 expression intensity) which we termed CD8int and CD8hi. When analyzed by flow-cytometry, ex vivo generated CD8int, but not CD8hi, expressed high levels of CD69, PD-L1 and CD44. In contrast, SP CD8 T cells developed in the presence of rIL7 did not upregulate these markers. Since IL7 promotes survival and proliferation of TCR-triggered DPs while IL4 affects their differentiation, we admixed both cytokines during the co-culture and found a dominant rIL4 effect: the phenotype of SP CD8 T cells was similar to that induced by rIL4 alone. Taken together, our findings demonstrate that some DP thymocytes are efficiently selected in our system by OTI-specific positively selecting peptides. Notably, the addition of rIL7 leads to the development and maturation of classic CD8 T cells whereas rIL4 induces both classic and innate CD8 T cells.This work was supported by grant a from CIHR. Disclosures:No relevant conflicts of interest to declare.

Suppression of antigen-specific CD4+ T cell activation by SRA/CD204 through reducing the immunostimulatory capability of antigen-presenting cell

Pattern recognition scavenger receptor SRA/CD204, primarily expressed on specialized antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer's disease, endotoxic shock, host defense, and cancer development. SRA/CD204 was also recently shown to function as an attenuator of vaccine response and antitumor immunity. Here, we, for the first time, report that SRA/CD204 knockout (SRA(-/-)) mice developed a more robust CD4(+) T cell response than wild-type mice after ovalbumin immunization. Splenic DCs from the immunized SRA(-/-) mice were much more efficient than those from WT mice in stimulating naïve OT-II cells, indicating that the suppressive activity of SRA/CD204 is mediated by DCs. Strikingly, antigen-exposed SRA(-/-) DCs with or without lipopolysaccharide treatment exhibited increased T-cell-stimulating activity in vitro, which was independent of the classical endocytic property of the SRA/CD204. Additionally, absence of SRA/CD204 resulted in significantly elevated IL12p35 expression in DCs upon CD40 ligation plus interferon gamma (IFN-γ) stimulation. Molecular studies reveal that SRA/CD204 inhibited the activation of STAT1, mitogen activated protein kinase p38, and nuclear factor-kappa B signaling activation in DCs treated with anti-CD40 antibodies and IFN-γ. Furthermore, splenocytes from the generated SRA(-/-) OT-II mice showed heightened proliferation upon stimulation with OVA protein or MHC-II-restricted OVA(323-339) peptide compared with cells from the SRA(+/+) OT-II mice. These results not only establish a new role of SRA/CD204 in limiting the intrinsic immunogenicity of APCs and CD4(+) T cell activation but also provide additional insights into the molecular mechanisms involved in the immune suppression by this molecule.

Enhanced IL-10 production by CD4+T cells primed in IL-15Rα-deficient mice

In this study, we investigated the functional outcomes of CD4(+) T cells primed in the absence of IL-15 transpresentation. Compared with their WT counterparts primed in WT mice, IL-15Rα KO CD4(+) T cells primed in KO mice were found to exclusively overproduce IL-10 upon in vitro restimulation(.) The comparable expression of IL-4 and Foxp3 in CD4(+) T cells primed in the WT and IL-15Rα KO mice indicated that this was neither due to T(H) 2- nor Treg cell-differentiation. IL-10 overproduction was also observed when OVA-specific TCR transgenic CD4(+) T (OT-II) cells were primed in KO mice, excluding an intrinsic deficiency of KO CD4(+) T cells. To investigate the WT and KO microenvironment, DCs from both WT and IL-15Rα KO mice were compared. DCs from both backgrounds were indistinguishable in their steady-state survival and in their expression of MHC class II and costimulatory molecules CD80, CD86, and CD40. However, IL-15Rα KO DCs primed OT-II cells in vitro to produce higher levels of IL-10 upon their restimulation. Additionally, IL-15Rα KO DCs produced significantly more IL-10 upon activation, and IL-10 neutralization during DC-mediated in vitro priming abolished IL-10 overproduction by CD4(+) T cells. Thus, IL-15Rα KO DCs provide an IL-10-enriched environment that preferentially primes CD4(+) T cells for more IL-10 production, highlighting a regulatory role for IL-15 transpresentation in CD4(+) T-cell priming.

Helios Is Associated with CD4 T Cells Differentiating to T Helper 2 and Follicular Helper T Cells In Vivo Independently of Foxp3 Expression

BackgroundAlthough in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized.FindingsHelios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen-matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-γ - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-κB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA.ConclusionsHelios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain.

Selective effects of NF‐κB1 deficiency in CD4+ T cells on Th2 and TFh induction by alum‐precipitated protein vaccines

NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.