OT-I Mice Research Articles

DIPG-17. CD155 regulates cell growth and immune evasion in diffuse intrinsic pontine glioma

There is an unmet need for more effective treatment strategies for diffuse intrinsic pontine glioma (DIPG), a devastating brain tumour arising in children and young adults. While immunotherapy is emerging as a powerful approach to treatment of other cancers, clinical trials with immune checkpoint inhibitors have failed to show a survival benefit for DIPG patients. In this study, we analysed the expression of immune checkpoint molecules on the surface of human and murine DIPG cells by flow cytometry and identified CD155 and B7-H3 as the most highly expressed checkpoint molecules, with minimal expression of PD-L1, PD-L2, Galectin-9, CEACAM-1, CD86, CD252 and CD137. These findings were confirmed in primary patient samples from pediatric brain tumours, including high-grade gliomas, medulloblastomas and ependymomas. To test whether CD155 inhibition increases susceptibility to CD8+ T cell killing in vitro, we cultured DIPG cells expressing ovalbumin (OVA) with CD8+ T cells from OT-I mice, which express T cell receptors specific for OVA. Addition of CD155 blocking antibodies to these cultures increased expression of T cell activation markers (CD25, CD44 and CD69) as well as T cell-mediated tumour killing, supporting the notion that CD155 can function as an immune checkpoint in DIPG. In addition to its effects on T cells, CD155 also exerted direct effects on tumour cells: treatment with anti-CD155 antibodies led to impaired cell viability, and shRNA-mediated knockdown of CD155 resulted in reduced cell proliferation in vitro. Strikingly, knockdown of CD155 also led to reduced growth of DIPG cells in vivo, and mice transplanted with the CD155-deficient cells had a clear survival benefit compared to mice transplanted with wild type cells. These studies demonstrate that CD155 functions as an immune checkpoint and as a regulator of tumor growth in DIPG, and suggest that targeting CD155 could be a valuable therapeutic strategy for this devastating disease.

Loss of the nuclear receptor PPARγ primes CD8+ T cells for exhaustion

Abstract Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to nuclear hormone superfamily, with three distinct types identified: PPARapha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). The role of PPARγ in the immune system has been mainly studied in macrophages, where it is known to mediate M2 polarization. Synthetic ligands for activating PPARγ ameliorate disease outcomes in various models of inflammation and autoimmunity. Here, we investigated how PPARγ affects antigen-specific effector and memory responses of CD8+ T cells using OT-I TCR transgenic mice and OT-I mice with T cell-specific PPARγ deletion. During CD8+ T effector differentiation in vitro by IL-2 culture following stimulation with cognate antigen, PPARγ deletion resulted in a rapid and enhanced T effector cell expansion and activation. However, PPARγ deficient OTI cells were more prone to exhaustion. This effect coincided with decreased mitochondrial fitness and spare respiratory capacity, high ROS levels and a significant increase in oxidized lipids. During generation of T effector cells in vivo, PPARγ deletion resulted in similar outcomes. At the memory phase there were no numerical differences in the T memory subsets in vivo, but upon rechallenge in vitro, PPARγ-deficient antigen-specific T cells had impaired capacity for effector differentiation. Our findings uncover a new role of PPARγ in CD8+ T responses and indicate that PPARγ has an indispensable role in regulating metabolism-mediated cues that imprint T cell differentiation and long-term effector capacity. Supported by RO1CA212605, RO1CA229784-01, RO1CA238263 (to VAB)

Tregs suppress antigen-specific CD8+ T cells in vivo by depleting pMHC-I complexes from Dendritic Cells

Abstract T Regulatory cells (Tregs) regulate antigen-specific immune responses by using a variety of mechanisms. Recent studies have shown that antigen-specific Treg can suppress CD4+ effector T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. It remains unclear as to how Treg suppress CD8+ T effectors, particularly in vivo. To explore Treg-mediated suppression of CD8+ T cells, we generated CD4+ iTregs from OT-II mice specific for Ova323–339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257–264 (SIINFEKL) in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of DCs pulsed with both Ova323–339 and Ova257–264 or with DCs singly pulsed with each peptide. Interestingly, the expansion of OT-I CD8+ T cells in vivo was not suppressed by OT-II iTreg when the two peptides were presented on separately pulsed DCs, but was suppressed when both peptides were presented on the same DCs. OT-II Tregs depleted the Kb-SIINFEKL complexes from the DC surface in vitro by a process of trogocytosis only in the presence of their cognate antigen. The uptake of Kb-SIINFEKL complexes by OT-II Treg was not secondary to leakage of free SIINFEKL from the DC surface as OT-II Treg from MHC-I deficient mice were as efficient in uptake of pMHC-I as Treg from wild type mice. Multiphoton imaging microscopy in vivo reveals a close interaction between Tregs and CD8+ T cells suggesting that suppression of CD8+ T cells in vivo requires close proximity between Treg and responder CD8+ T cell may involve an artificial synapse between Treg and CD8+ T cell.

A Phenotypic Analysis of Involucrin–Membrane-Bound Ovalbumin Mice after Adoptive Transfer of Ovalbumin-Specific CD8+ T Cells

To investigate the mechanism of autoimmunity and peripheral tolerance in the skin, several transgenic mouse strains expressing membrane-bound ovalbumin (mOVA) as an epidermal self-antigen under the control of keratinocyte-specific promotors, such as keratin 5 and keratin 14, were employed in combination with adoptive transfer of CD8+ T cells from OT-I mice (OT-I T cells) that recognize an ovalbumin-derived peptide. However, these strains showed bodyweight loss and required additional inflammatory stimuli, such as γ-irradiation and tape-stripping, to induce skin inflammation. In this study, we generated a mouse strain expressing mOVA under the control of human involucrin promoter (involucrin-mOVA mice). In contrast to previous strains, involucrin-mOVA mice spontaneously developed skin inflammation after the transfer of OT-I T cells in the absence of external stimuli without significant bodyweight loss. We focused on the skin infiltration process of OT-I T cells and found that transferred OT-I T cells accumulated around the hair follicles in the early phase of skin inflammation, and in the later phase, the skin inflammation spontaneously resolved despite the remaining OT-I T cells in the skin. Our involucrin-mOVA mice will provide a promising tool to investigate the pathogenesis and the tolerance mechanisms of cytotoxic skin autoimmunity.

P064 IMU-838, a Small Molecule DHODH Inhibitor in Phase 2 Clinical Trial for Ulcerative Colitis, Shows Potent Anti-inflammatory Activity in Cell-Culture-Based and In Vivo Systems

Abstract Background IMU-838 (vidofludimus calcium) is a well-tolerated and orally available small molecule inhibitor of dihydroorotate dehydrogenase (DHODH) and is currently in phase 2 clinical development for ulcerative colitis (UC). Here, we investigated the effect of IMU-838 on different immune cell subsets and analysed the mechanism of action in more detail in T cells. Methods The effect of IMU-838 on regulatory macrophages (Mregs) was investigated by adding IMU-838 or anti-TNFα antibodies (infliximab, adalimumab), alone or in combination, in a mixed lymphocyte reaction assay, determining Mregs (CD206+CD14+) by FACS analysis. To investigate the effect of IMU-838 on human T and B cells, PBMC were stimulated with PHA (48h) or Oligonucleotide 2006-PTO (70h), respectively. An affinity-dependent effect of IMU-838 was assessed on CD8 T cells from OT-I and OT-III mice, containing a high and a low affinity T cell receptor (TCR), respectively, for OVA257-264 peptide. These cells were stimulated with ovalbumin (OVA) peptide loaded splenocytes or anti-CD3/anti-CD28 for 3 days. Oxidative phosphorylation (OXPHOS) and glycolysis in murine T cells was investigated by the Seahorse/Agilent technique in murine CD4 and CD8 T cells activated with anti-CD3/anti-CD28 with or without IMU-838. Results IMU-838 slightly induced Mregs, but strongly and dose-dependently decreased TNFα and IL-6 secretion. Anti-TNFα antibodies alone strongly induced Mregs, but also increased IL-6 levels. When IMU-838 was added, Mregs induction was even higher, and IL-6 was reduced. Besides reducing proinflammatory cytokines secreted by macrophages, IMU-838 also inhibits proliferation of activated B and T cells, as well as inflammatory T cell cytokine secretion, IL-17A, IL-17F and IFNγ, and increased apoptosis up to 3-fold compared to vehicle control. By investigating IMU-838’s role on affinity related activity status of T cells, it was shown that IMU-838 strongly inhibited cell proliferation in the peptide stimulated high affinity, but not in the low affinity, CD8 T cells. Peptide stimulated high affinity T cells show an upregulation of OXPHOS and glycolysis pathways, which are involved in the generation of the cell’s energy supply, compared to low affinity cells. IMU-838 strongly inhibited OXPHOS and glycolysis in both CD4 and CD8 T cells. Conclusion IMU-838 reduces the proinflammatory immune cell response by inducing Mregs, reducing pro-inflammatory cytokine secretion and reducing immune cell proliferation. DHODH is only important in cells that received a strong stimulus and are highly metabolically active. Therefore, IMU-838 will only specifically target these cells and will still allow for a normal immune response, representing a huge safety advantage for treatment of IBD patients.

Rapid video-based deep learning of cognate versus non-cognate T cell-dendritic cell interactions

Identification of cognate interactions between antigen-specific T cells and dendritic cells (DCs) is essential to understanding immunity and tolerance, and for developing therapies for cancer and autoimmune diseases. Conventional techniques for selecting antigen-specific T cells are time-consuming and limited to pre-defined antigenic peptide sequences. Here, we demonstrate the ability to use deep learning to rapidly classify videos of antigen-specific CD8+ T cells. The trained model distinguishes distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and DCs over 20 to 80 min. The model classified high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalized well to other types of high and low affinity CD8+ T cells. The classification accuracy achieved by the model was consistently higher than simple image analysis techniques, and conventional metrics used to differentiate between cognate and non-cognate T cells, such as speed. Also, we demonstrated that experimental addition of anti-CD40 antibodies improved model prediction. Overall, this method demonstrates the potential of video-based deep learning to rapidly classify cognate T cell-DC interactions, which may also be potentially integrated into high-throughput methods for selecting antigen-specific T cells in the future.

Prosurvival IL-7-Stimulated Weak Strength of mTORC1-S6K Controls T Cell Memory via Transcriptional FOXO1-TCF1-Id3 and Metabolic AMPKα1-ULK1-ATG7 Pathways.

CD8+ memory T (TM) cells play a critical role in immune defense against infection. Two common γ-chain family cytokines, IL-2 and IL-7, although triggering the same mTORC1-S6K pathway, distinctly induce effector T (TE) cells and TM cells, respectively, but the underlying mechanism(s) remains elusive. In this study, we generated IL-7R-/and AMPKα1-knockout (KO)/OTI mice. By using genetic and pharmaceutical tools, we demonstrate that IL-7 deficiency represses expression of FOXO1, TCF1, p-AMPKα1 (T172), and p-ULK1 (S555) and abolishes T cell memory differentiation in IL-7R KO T cells after Listeria monocytogenesis rLmOVA infection. IL-2- and IL-7-stimulated strong and weak S6K (IL-2/S6Kstrong and IL-7/S6Kweak) signals control short-lived IL-7R-CD62L-KLRG1+ TE and long-term IL-7R+CD62L+KLRG1- TM cell formations, respectively. To assess underlying molecular pathway(s), we performed flow cytometry, Western blotting, confocal microscopy, and Seahorse assay analyses by using the IL-7/S6Kweak-stimulated TM (IL-7/TM) and the control IL-2/S6Kstrong-stimulated TE (IL-2/TE) cells. We determine that the IL-7/S6Kweak signal activates transcriptional FOXO1, TCF1, and Id3 and metabolic p-AMPKα1, p-ULK1, and ATG7 molecules in IL-7/TM cells. IL-7/TM cells upregulate IL-7R and CD62L, promote mitochondria biogenesis and fatty acid oxidation metabolism, and show long-term cell survival and functional recall responses. Interestingly, AMPKα1 deficiency abolishes the AMPKα1 but maintains the FOXO1 pathway and induces a metabolic switch from fatty acid oxidation to glycolysis in AMPKα1 KO IL-7/TM cells, leading to loss of cell survival and recall responses. Taken together, our data demonstrate that IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via activation of transcriptional FOXO1-TCF1-Id3 and metabolic AMPKα1-ULK1-ATG7 pathways. This (to our knowledge) novel finding provides a new mechanism for a distinct IL-2/IL-7 stimulation model in T cell memory and greatly impacts vaccine development.

P08.02 Harnessing T cells to target brain metastasis

Abstract BACKGROUND Up to 60% of melanoma patients develop brain metastases (BrM). These patients have a poor prognosis and limited treatment options. Immune checkpoint inhibitors (ICI) targeting Cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and Programmed cell death protein-1 (PD-1) have revolutionized the treatment of melanoma and their efficacy has been also demonstrated in melanoma BrM. Our group previously demonstrated that ICI (combined α-PD-1 and α-CTLA-4) enhances chemokine-dependent infiltration of cytotoxic T lymphocytes (CTLs) into melanoma BrMs in preclinical models, accompanied by upregulated expression of T cell attracting chemokines in tumours. Notably, CTLs infiltrating BrM expressed only some of the chemokine receptors (CRs) interacting with ICI-induced chemokines in BrM, providing a rationale to over-express the “missing” CRs in T cells to enhance their homing to tumours in the context of adoptive T cell therapy (ACT). MATERIALS AND METHODS OT-I cells were isolated from OT-I mice and differentiated ex vivo into effector (TEF) and memory (TCM) CD8+ T cells. Tumour infiltrating lymphocytes (TILs) from B16 tumour-bearing mice treated with ICI were isolated using magnetic beads, activated and expanded ex vivo. Expression of CRs and activation markers in ex vivo cultured T cells were quantified by qPCR and/or flow cytometry. The migration of human blood CD8+ T cells towards chemokines of interest were measured in ex vivo migration assays. RESULTS The same CRs that were missing on BrM-infiltrating CTLs in vivo models were also absent from OT-I TEF (CCR7low/CD44high/CD62Llow) and TCM (CCR7high/CD44low/CD62Lhigh) cells, as well as from TILs expanded ex vivo for use in ACT. Furthermore, we observed no increase in migration of human T cells towards chemokines interacting with the “missing” CRs in comparison to the baseline migration, suggesting that these CRs are also absent from human T cells. CONCLUSION Ex vivo expanded T cells that are used in ACT are missing several CRs that are interacting with chemokines upregulated in BrM. We hypothesise that the use of genetically engineered T cells expressing the “missing” CRs in ACT has the potential to enhance ACT efficacy in combination with ICI.

CXCR6 is required for antitumor efficacy of intratumoral CD8+ T cell

BackgroundIncreasing infiltration of CD8+ T cells within tumor tissue predicts a better prognosis and is essential for response to checkpoint blocking therapy. Furthermore, current clinical protocols use unfractioned T cell...

Abstract 1579: Oncolytic adenovirus Delta-24-RGDOX collaborates with adoptive cell therapy to sustain anti-tumor immunity

Abstract Oncolytic virotherapy and adoptive T cell therapy (ACT) are promising strategies for cancer treatment, but both are underdeveloped into routine clinical interventions for solid tumors. We reported previously that localized treatment with oncolytic adenovirus Delta-24-RGDOX (DNX-2440), which expresses immune co-stimulator OX40 ligand, stimulated tumor-specific T cells proliferation and migration to untreated disseminated tumors. We hypothesize that intratumorally injected Delta-24-RGDOX complements with ACT to overcome antigen escape, leading to more sustainable anti-cancer effect. To test the hypothesis, we used B16-OVA-C57BL/6 subcutaneous (s.c.)/s.c. melanoma model to assess the effect of the combination therapy. Briefly, 10 days after the first tumor implantation, we injected OVA-specific CD8+ T cells from OT-I mice into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. We found the combination was more potent to inhibit the injected 1st tumor and untreated disseminated 2nd tumor growth and resulted in improved survival rate than either of the agent alone (p less than 0.05). Interestingly, we observed relapse of the regressed tumors in the group treated by T cells alone, which recapitulated the effect of CAR T therapy in cancer patients. But the relapse was not observed in most of the mice from the combination group, indicating the combination induces more sustainable tumor regression than T cells alone. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory against the same tumor cells. Furthermore, profiling the tumor-infiltrating lymphocytes with flow cytometry revealed that the combination promotes the T cell presence within the tumor and increase the population of the T cells targeting other antigens in addition to the original ACT targeted antigen. Collectively, the results demonstrate that Delta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to more sustainable tumor regression and improved survival rate. Our study suggests the virus induces antigen spread, resulting in T cell repertoire to target more tumor-associated antigens to overcome cancer relapse due to cancer heterogeneity and limited targets in ACT and CAR T therapy. Citation Format: Hong Jiang, Teresa T. Nguyen, Dong Ho Shin, Sagar S. Sohoni, Sumit Gupta, Yanhua Yi, Xuejun Fan, Candelaria Gomez-Manzano, Juan Fueyo. Oncolytic adenovirus Delta-24-RGDOX collaborates with adoptive cell therapy to sustain anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1579.

Platelet-Targeted Gene Transfer Induces Immune Tolerance in a Primed Model

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) is effective in inducing immune tolerance not only to the coagulation factor VIII and IX, but also to the non-coagulant protein ovalbumin (OVA) in a model without pre-existing immunity (the non-primed model). In the present study, we explored whether platelet-specific gene transfer could induce immune tolerance in the primed model. To address this question, we first used C57BL6 wild type (WT) mouse models. Both donors and recipients were immunized with OVA to induce anti-OVA antibody development. OVA expression was introduced by 2bOVA lentivirus (LV) transduction of hematopoietic stem cells (HSCs) from WT/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with a sub-lethal 6.6Gy total body irradiation (TBI). 2bGFP was used as a control vector in parallel. Animals were analyzed after at least 4 weeks of bone marrow (BM) reconstitution. The viability of OVA genetically modified HSCs was confirmed by PCR in all 2bOVA-transduced recipients. After full bone marrow constitution, which takes about 12 weeks, the engraftment in the 2bOVA group (87.4±4.4%, n=5) was similar to that in the 2bGFP group (87.8±3.9%, n=5) as determined by flow cytometry. The OVA protein level was 6.7±2.8 ng/108platelets (n=5) in 2bOVA transduced recipients. Anti-OVA antibody titers declined with time in both groups. Six months after transplantation, recipients were rechallenged with OVA. All of the anti-OVA titers in 2bGFP-transduced recipients (n=5) increased dramatically, while only one in the 2bOVA group slightly increased. Normalized anti-OVA antibody titers in the 2bOVA group (90.8±152.7) were significantly lower than those in the 2bGFP group (1278±2101) after OVA rechallenge, demonstrating that platelet-specific OVA gene delivery to HSCs can suppress anti-OVA immune responses in OVA-primed WT mice.To explore how immune suppression is established after platelet-specific gene transfer in the OVA-primed model, we transduced HSCs from OVA-specific TCR transgenic mice (OTII/CD45.2, in which greater than 97% CD4 T+ cells are OVA-specific) with 2bOVALV and transplanted into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy TBI. Untransduced transplanted mice were used as a control. After BM reconstitution, the OVA protein level was 32.3±6.3 ng/108 platelets (n=5) in the 2bOVA-transduced group. Anti-OVA antibody titers in both groups declined with time. When all the recipients were rechallenged with OVA, 2 of 3 anti-OVA titers in the untransduced group increased, while none of the titers in the 2bOVA group increased. Normalized anti-OVA antibody titers in the 2bOVA group (4.1±4.9) were significantly lower than those in the un-transduced group (152.3±214.5), demonstrating that platelet-specific OVA gene delivery to HSCs can also suppress the anti-OVA immune response in the OVA-primed WT mice that received 2bOVA-transduced HSCs from OTII mice (the OTII/WT model). After BM reconstitution, the engraftments in the 2 groups were similar (81.4±2.6%, and 80.9±2.5%, respectively), but donor-derived CD45.2+CD4+ (OTII/CD4) T cells in the 2bOVA (0.2±0.1%) group were consistently significantly lower than in the untransduced group (4.2±1.1%) in peripheral blood during the entire study course. Also, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA group compared to the untransduced group. Interestingly, there were no differences in either percentage or total cell number of CD45.2+CD4+ T cells in the thymus between the 2 groups, indicating that central tolerance may not play a role in the immune tolerance induced after platelet-targeted gene therapy. Meanwhile, the frequency and total number of endogenous CD4+ T cells were similar in the 2 groups. Of note, the percentages of donor-derived regulatory T (Treg) cells in the 2bOVA group were significantly higher than those in the untransduced group in peripheral blood, spleen, and lymph nodes, while there were no significant differences in the total numbers of donor-derived Treg cells between the 2 groups.Taken together, our studies demonstrate that platelet-specific gene therapy can induce immune tolerance in a primed system through two distinct pathways, peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. DisclosuresNo relevant conflicts of interest to declare.

Tolerization of CD4+ Recent Thymic Emigrants Is Required to Prevent RBC-Specific Autoimmunity

BackgroundLoss of humoral tolerance to red blood cell (RBC)-specific antigens may lead to the development of pathogenic autoantibodies and result in autoimmune hemolytic anemia, a severe and sometimes fatal disease. Recently, it has been demonstrated that although RBC-specific autoreactive B cells escape central tolerance (and can secrete autoantibodies), stringent T cell tolerance is a stopgap to autoimmunity; however, T cell tolerance mechanisms utilized against RBC antigens is largely unknown.Thymic central tolerance is a process by which newly-formed autoreactive T cells undergo negative selection by deletion, anergy, or diversion into a regulatory T cell (Treg). This process ensures that newly-minted T cells are self-tolerant and functional. Elimination of >95% of thymocytes occurs during central tolerance. However, while rigorous, central tolerance sometimes fails and autoreactive T cells escape to the periphery. The youngest peripheral T cells, recent thymic emigrants (RTEs), are functionally distinct from mature T cells and are more susceptible to peripheral tolerance mechanisms in response to autoantigens. Herein, we assess central and peripheral T tolerance to RBC-specific autoantigens.MethodsHOD transgenic mice express an RBC-specific fusion protein consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and human blood group, Duffy (D). To assess tolerance, HOD mice were bred with OTII mice, CD4+ T cell receptor (TCR) transgenic mice with specificity for OVA (contained within HOD). HODxOTII F1s were immunized with 100ug of OVA/CFA. To assess central tolerance, thymi from 8-12 week old HODxOTII F1 mice were harvested and stained with antibodies to identify OTIIs and Tregs. For RTE analysis, HOD mice were bred with OTII.Rag2p-GFP (referred to as HODxRTE mice), OTII T cells that express GFP during TCR rearrangement. GFP+ OTII RTEs were stained with antibodies against CD73, FR4, and PD1. To assess function, GFP+ OTII RTEs were sorted and 3x105 RTEs were infused into naïve B6 mice. Recipient mice received a 100uL packed HOD RBC transfusion and RTE proliferation was assessed at 3 days by Ki67 staining.ResultsHOD+OTII+ mice had profound tolerance to the HOD RBC autoantigen; immunization with OVA/CFA elicited strong antibody responses in HOD-OTII+ littermates, but not autoreactive HOD+OTII+ mice. To determine whether OTIIs underwent central tolerance, thymi were analyzed. No significant differences in total numbers of thymocytes or CD4+ OTIIs was observed in autoreactive HOD+OTII+ mice, compared to control HOD-OTII+. Additional phenotyping for co-expression of CD25 and FoxP3 (a phenotype consistent with Tregs) revealed no difference, suggesting autoreactive OTII T cells were not diverted into a regulatory program. Thus, negative selection of RBC autoreactive CD4+ T cells does not occur in the thymus.Given that RTEs are subject to tolerization in response to peripherally expressed autoantigens, HODxRTE F1s were used to assess whether RBC-specific autoreactive OTIIs are tolerized as RTEs. RTEs were divided into GFPhi (< 1 week in the periphery), GFPint (2-3 weeks in the periphery), and GFPneg (mature T cells). In HOD+RTE+ mice, GFPhi RTEs had significantly higher levels of PD1, a marker of exhaustion, compared to controls. As RTEs matured, PD1 expression slightly decreased, but remained high compared to controls. Additionally, as autoreactive OTII RTEs matured, a significant number OTIIs expressed CD73hiFR4hi, a phenotype consistent with anergy. On average, 5% of GFPneg OTIIs from HOD-RTE+ mice expressed CD73hiFR4hi, compared to over 60% in autoreactive HOD+RTE+ mice. To assess function, sorted RTEs from HODxRTE mice were adoptively transferred into naïve mice and assessed for proliferation post HOD RBC transfusion. All RTEs sorted from HOD-RTE+ mice proliferated (>95% of OTIIs were Ki67+). However, RTEs derived from HOD+RTE+ mice had diminished proliferative capacity upon maturation (Ki67+ RTEs: GFPhi 65%, GFPint 35%, GFPneg 15%).ConclusionsThese data demonstrate that RBC-specific autoreactive CD4+ T cells are neither deleted nor diverted into a Tregs during thymic central tolerance, rather peripheral tolerance mechanisms occurring during RTE maturation is required. Together, these data provide insight into RBC-specific T cell tolerance mechanisms, pinpoint when they occur, and demonstrate that if RTE maturation does not occur, RBC autoimmunity may ensue. DisclosuresNo relevant conflicts of interest to declare.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis. Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Pro-survival IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via transcriptional FOXO1 and metabolic AMPK pathways

Abstract Distinct strengths of stimuli (DSS) is a well-known model for T cell memory. Two common γ-chain (γc)-family cytokines IL-2 and IL-7, which trigger the same mTORC1-S6K pathway, distinctly induce effector T (Te) cells and memory T (Tm) cell differentiation. However, their underlying mechanisms are elusive. In this study, we want to elucidate molecular mechanism for IL-7-induced Tm (IL-7/Tm) cells by performing in vitro and in vivo experiments using genetic and pharmacological tools. We show that IL-2 and IL-7 stimulate strong and weak S6K signals leading to IL-7R−CD62L−KLRG1+ Te and IL-7R+CD62L+KLRG1− Tm cell formation in our study model of adoptive transfer of T cells derived from wild-type and IL-7R knockout (KO)/OTI mice and L. monocytogenes rLmOVA infection. We further show that IL-7/Tm cells upregulate transcriptional factors (FOXO1, Id3 and TCF1) for regulating Tm’s differentiation and promote expression of autophagy/metabolic molecules (AMPK, ULK1 and ATG7) for controlling Tm’s fatty acid oxidation (FAO) metabolism by flow cytometry, Western blotting, confocal and electron microscopy and Seahorse assay analyses, and vice versa for IL-2-induced Te (IL-2/Te) cells. IL-7/Tm cells derived from AMPK KO/OTI mice abolish expression of ULK1 and ATG7, FAO metabolism and long-term survival, while IL-2/Te cells with S6K deficiency show Tm’s differentiation, metabolic/transcriptional pathways and prolonged survival. Taken together, our data demonstrate that pro-survival IL-7-stimulated weak S6K signaling controls T cell memory via transcriptional FOXO1 and metabolic AMPK pathways. The novel finding provides a new potential mechanism for DSS model in T cell memory and has a great impact on vaccine development and immunotherapy.

Regulatory T cells suppress antigen-specific CD8+ T cells in vivo by modulating the immune-synapse

Abstract T Regulatory cells (Tregs) plays an important role in regulating self and foreign antigens related immune response by different mechanisms. Recent studies have shown that antigen-specific Treg suppress CD4+ effectors T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. These, results raise questions as to how Treg suppress CD8+ T effectors, particularly in vivo. To explore Treg mediated suppression of CD8+ T cells, we generated CD4+ iTregs from OT-II mice specific for Ova323–339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257–264 (SIINFEKL) in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of dendritic cells (DCs) pulsed with both Ova323–339 and Ova257–264 or with DCs singly pulsed with each peptide. Surprisingly, the expansion of OT-I CD8+ T cells in vivo was not suppressed by OT-II iTreg when the two peptides were presented on separately pulsed DCs, but was suppressed when both peptides were presented on the same DCs. OT-II Tregs depleted the Kb-SIINFEKL complex from the DC surface by a process of trogocytosis in vitro only in presence of their cognate antigen. Imaging experiments in vivo depict a close interaction between Tregs and CD8+ T cells suggesting that suppression of CD8+ T cells in vivo requires close proximity between Treg and responder CD8+ T cell and that pMHCI and pMHCII complexes may be closely associated in the immune synapse.

Crassolide Suppresses Dendritic Cell Maturation and Attenuates Experimental Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the production of β2-glycoprotein I (β2GPI)-dependent autoantibodies, with vascular thrombosis or obstetrical complications. Around 20% of APS patients are refractory to current treatments. Crassolide, a cembranoid diterpene extracted from soft corals, is a potential therapeutic candidate. Here, to examine the anti-inflammatory properties of crassolide, we first determined its effects on bone marrow-derived and splenic dendritic cells (DC). Specifically, we applied lipopolysaccharide (LPS) or β2GPI stimulation and measured the expressions of CD80 and CD86, and secretions of cytokines. We also determined in the OT-II mice, if bone marrow-derived DC was able to stimulate antigen-specific T cells. Moreover, we examined the therapeutic potential of crassolide postimmunization in a murine model of APS that depended on active immunization with β2GPI. The vascular manifestations were evaluated in terms of fluorescein-induced thrombi in mesenteric microvessels, whereas the obstetric manifestations were evaluated based on the proportion of fetal loss after pregnancy. We also measured blood titers of anti-β2GPI antibody, splenic cell proliferative responses and cytokine secretions after β2GPI stimulation ex vivo. Finally, we determined in these mice, hematological, hepatic and renal toxicities of crassolide. Crassolide after LPS stimulation suppressed DC maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12 and IL-23, and downstream T cell activation. Crassolide could partially ameliorate both the vascular and obstetric manifestations of APS in BALB/c mice. Both blood titers of anti-β2GPI antibody and splenic cell proliferation after β2GPI stimulation were reduced. Splenic Th1 and Th17 responses were also lowered after β2GPI stimulation. Finally, within therapeutic doses of crassolide, we found no evidence of its toxicity. In conclusion, we showed the ability of crassolide to suppress DC and downstream T cell responses. Crassolide is therefore a potential candidate for adjunctive therapy in APS.

GILT in tumor cells improves T cell-mediated anti-tumor immune surveillance

The lysosomal thiol reductase GILT catalyzes the reduction of disulfide bonds of protein antigens, facilitating antigen-presenting cells (APCs) to present antigen to T cells. However, whether GILT expression in tumor cells can be associated with improved T cell-mediated anti-tumor responses remains unknown. Here, we identify that GILT is able to facilitate anti-tumor immune surveillance via promoting MHC class I mediated-antigen presentation in colon carcinoma. By using mice model bearing colon tumors, we find that GILT inhibites tumor growth in vivo with more leucocytes infiltration but has no effect on tumor cell development in vitro in terms of proliferation, cell cycle and migration. Furthermore, by using transgenic OT-I mice, we recognize the tumor-expressing OVA peptide, a surrogate tumor antigen, we find that GILT is capable of enhancing MHC class I mediated antigen presentation and improving specific CD8+ T cell anti-tumor responses in murine colon carcinoma. These findings propose the boost of GILT-MHC-I axis in tumors as a viable option for immune system against cancer.

Isolevuglandin-Modified Cardiac Proteins Drive CD4+ T-Cell Activation in the Heart and Promote Cardiac Dysfunction.

Despite the well-established association between T-cell-mediated inflammation and nonischemic heart failure, the specific mechanisms triggering T-cell activation during the progression of heart failure and the antigens involved are poorly understood. We hypothesized that myocardial oxidative stress induces the formation of isolevuglandin (IsoLG)-modified proteins that function as cardiac neoantigens to elicit CD4+ T-cell receptor (TCR) activation and promote heart failure. We used transverse aortic constriction in mice to trigger myocardial oxidative stress and T-cell infiltration. We profiled the TCR repertoire by mRNA sequencing of intramyocardial activated CD4+ T cells in Nur77GFP reporter mice, which transiently express GFP on TCR engagement. We assessed the role of antigen presentation and TCR specificity in the development of cardiac dysfunction using antigen presentation-deficient MhcII-/- mice and TCR transgenic OTII mice that lack specificity for endogenous antigens. We detected IsoLG protein adducts in failing human hearts. We also evaluated the role of reactive oxygen species and IsoLGs in eliciting T-cell immune responses in vivo by treating mice with the antioxidant TEMPOL and the IsoLG scavenger 2-hydroxybenzylamine during transverse aortic constriction, and ex vivo in mechanistic studies of CD4+ T-cell proliferation in response to IsoLG-modified cardiac proteins. We discovered that TCR antigen recognition increases in the left ventricle as cardiac dysfunction progresses and identified a limited repertoire of activated CD4+ T-cell clonotypes in the left ventricle. Antigen presentation of endogenous antigens was required to develop cardiac dysfunction because MhcII-/- mice reconstituted with CD4+ T cells and OTII mice immunized with their cognate antigen were protected from transverse aortic constriction-induced cardiac dysfunction despite the presence of left ventricle-infiltrated CD4+ T cells. Scavenging IsoLGs with 2-hydroxybenzylamine reduced TCR activation and prevented cardiac dysfunction. Mechanistically, cardiac pressure overload resulted in reactive oxygen species-dependent dendritic cell accumulation of IsoLG protein adducts, which induced robust CD4+ T-cell proliferation. Our study demonstrates an important role of reactive oxygen species-induced formation of IsoLG-modified cardiac neoantigens that lead to TCR-dependent CD4+ T-cell activation within the heart.

Antigen- and Epitope-Delivering Nanoparticles Targeting Liver Induce Comparable Immunotolerance in Allergic Airway Disease and Anaphylaxis as Nanoparticle-Delivering Pharmaceuticals

The targeting of natural tolerogenic liver sinusoidal endothelial cells (LSEC) by nanoparticles (NPs), decorated with a stabilin receptor ligand, is capable of generating regulatory T-cells (Tregs), which can suppress antigen-specific immune responses, including to ovalbumin (OVA), a possible food allergen. In this regard, we have previously demonstrated that OVA-encapsulating poly(lactic-co-glycolic acid) (PLGA) nanoparticles eliminate allergic airway inflammation in OVA-sensitized mice, prophylactically and therapeutically. A competing approach is a nanocarrier platform that incorporates pharmaceutical agents interfering in mTOR (rapamycin) or NF-κB (curcumin) pathways, with the ability to induce a tolerogenic state in nontargeted antigen-presenting cells system-wide. First, we compared OVA-encapsulating, LSEC-targeting tolerogenic nanoparticles (TNPs) with nontargeted NPs incorporating curcumin and rapamycin (Rapa) in a murine eosinophilic airway inflammation model, which is Treg-sensitive. This demonstrated roughly similar tolerogenic effects on allergic airway inflammation by stabilin-targeting NPOVA versus nontargeted NPs delivering OVA plus Rapa. Reduction in eosinophilic inflammation and TH2-mediated immune responses in the lung was accompanied by increased Foxp3+ Treg recruitment and TGF-β production in both platforms. As OVA incorporates IgE-binding as well as non-IgE-binding epitopes, the next experiment explored the possibility of obtaining immune tolerance by non-anaphylactic T-cell epitopes. This was accomplished by incorporating OVA323-339 and OVA257-264 epitopes in liver-targeting NPs to assess the prophylactic and therapeutic impact on allergic inflammation in transgenic OT-II mice. Importantly, we demonstrated that the major histocompatibility complex (MHC)-II binding (former) but not the MHC-I binding (latter) epitope interfered in allergic airway inflammation, improving TNPOVA efficacy. The epitope-specific effect was transduced by TGF-β-producing Tregs. In the final phase of experimentation, we used an OVA-induced anaphylaxis model to demonstrate that targeted delivery of OVA and its MHC-II epitope could significantly suppress the anaphylaxis symptom score, mast cell release, and the late-phase inflammatory response. In summary, these results demonstrate comparable efficacy of LSEC-targeting versus pharmaceutical PLGA nanoparticles, as well as the ability of T-cell epitopes to achieve response outcomes similar to those of the intact allergens.

Threonyl-tRNA Synthetase Promotes T Helper Type 1 Cell Responses by Inducing Dendritic Cell Maturation and IL-12 Production via an NF-κB Pathway.

Threonyl-tRNA synthetase (TRS) is an aminoacyl-tRNA synthetase that catalyzes the aminoacylation of tRNA by transferring threonine. In addition to an essential role in translation, TRS was extracellularly detected in autoimmune diseases and also exhibited pro-angiogenetic activity. TRS is reported to be secreted into the extracellular space when vascular endothelial cells encounter tumor necrosis factor-α. As T helper (Th) type 1 response and IFN-γ levels are associated with autoimmunity and angiogenesis, in this study, we investigated the effects of TRS on dendritic cell (DC) activation and CD4 T cell polarization. TRS-treated DCs exhibited up-regulated expression of activation-related cell-surface molecules, including CD40, CD80, CD86, and MHC class II. Treatment of DCs with TRS resulted in a significant increase of IL-12 production. TRS triggered nuclear translocation of the NF-κB p65 subunit along with the degradation of IκB proteins and the phosphorylation of MAPKs in DCs. Additionally, MAPK inhibitors markedly recovered the degradation of IκB proteins and the increased IL-12 production in TRS-treated DCs, suggesting the involvement of MAPKs as the upstream regulators of NF-κB in TRS-induced DC maturation and activation. Importantly, TRS-stimulated DCs significantly increased the populations of IFN-γ+CD4 T cells, and the levels of IFN-γ when co-cultured with CD4+ T cells. The addition of a neutralizing anti-IL-12 mAb to the cell cultures of TRS-treated DCs and CD4+ T cells resulted in decreased IFN-γ production, indicating that TRS-stimulated DCs may enhance the Th1 response through DC-derived IL-12. Injection of OT-II mice with OVA-pulsed, TRS-treated DCs also enhanced Ag-specific Th1 responses in vivo. Importantly, injection with TRS-treated DC exhibited increased populations of IFN-γ+-CD4+ and -CD8+ T cells as well as secretion level of IFN-γ, resulting in viral clearance and increased survival periods in mice infected with influenza A virus (IAV), as the Th1 response is associated with the enhanced cellular immunity, including anti-viral activity. Taken together, these results indicate that TRS promotes the maturation and activation of DCs, DC-mediated Th1 responses, and anti-viral effect on IAV infection.