Effector T-cell Differentiation Research Articles

PI3 kinase delta governs regulatory T-cell biology and thus confers protection against atherosclerosis progression in mice

Abstract Introduction and Purpose Atherosclerosis is a chronic inflammatory disease of arteries, critically involving innate and adaptive immune cells like macrophages as well as T and B lymphocytes. Macrophages are major drivers of disease through the ingestion of lipoproteins, foam cell formation, and secretion of inflammatory mediators. Although macrophages outnumber other leukocytes in atherosclerotic plaques, T and B cells can shape the course of disease by promoting or mitigating inflammatory responses. The phosphoinositide 3-kinase delta (PI3Kd) exerts a key role in the regulation of immune responses including activation, proliferation, differentiation, and effector function of lymphocytes. Therefore, PI3Kd may represent a promising target for the modulation of inflammatory diseases. Consequently, we aimed to analyse the role of PI3Kd during atherogenesis. Methods and Results To investigate the role of haematopoietic PI3Kd in atherosclerosis, bone marrow from PI3Kd-/- or PI3Kd+/+ mice was transplanted into Ldlr-/- mice. After a 6-weeks-challenge by high fat diet, PI3Kd-/- recipient Ldlr-/- mice displayed profoundly impaired CD4+ and CD8+ T-cell numbers, CD4+ T-cell activation, CD4+ effector T-cell differentiation, and proatherogenic CD4+ T-helper (Th) 1 responses in para-aortic lymph nodes and spleen compared with PI3Kd+/+ transplanted controls. Surprisingly, the net effect of PI3Kd deficiency was a substantial increase of aortic inflammation and atherosclerosis in Ldlr-/- mice. Whereas plaque content and functions of macrophages including foam cell formation, efferocytosis, and cytokine secretion remained largely unaffected, haematopoietic PI3Kd ablation strongly reduced mature B cells and serum immunoglobulins in Ldlr-/- mice. Importantly, PI3Kd deficiency severely impaired numbers, proliferation, immunosuppressive functions, and stability of regulatory CD4+ T cells (Tregs). Consequently, adoptive transfer of PI3Kd+/+ Tregs fully restrained the atherosclerotic plaque burden in PI3Kd-/- transplanted Ldlr-/- mice without affecting B-cell numbers and serum immunoglobulins, whereas transferred PI3Kd-/- Tregs failed to relieve atherosclerosis progression. Conclusions Here, we demonstrate that PI3Kd plays a crucial role in Tregs, Th1 cells, and B cells during atherogenesis. Lack of PI3Kd signalling specifically in atheroprotective Treg responses outplays its impact on B-cell and proatherogenic Th1 responses, thus leading to aggravated atherosclerosis. Hence, PI3Kd is a key regulator of Treg biology and thereby protects against atherosclerosis, suggesting that fine-tuning PI3Kd signalling may represent a promising target for Treg-directed therapy.

M6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disorder in which excessive CD4+ T-cell activation and imbalanced effector T-cell differentiation play critical roles. Recent studies have implied a potential association between posttranscriptional N6-methyladenosine (m6A) modification and CD4+ T-cell-mediated humoral immunity. However, how this biological process contributes to lupus is not well understood. In this work, we investigated the role of the m6A methyltransferase like 3 (METTL3) in CD4+ T-cell activation, differentiation, and SLE pathogenesis both in vitro and in vivo.MethodsThe expression of METTL3 was knocked down and METTL3 enzyme activity was inhibited using siRNA and catalytic inhibitor, respectively. In vivo evaluation of METTL3 inhibition on CD4+ T-cell activation, effector T-cell differentiation, and SLE pathogenesis was achieved using a sheep red blood cell (SRBC)-immunized mouse model and a chronic graft versus host disease (cGVHD) mouse model. RNA-seq was performed to identify pathways and gene signatures targeted by METTL3. m6A RNA-immunoprecipitation qPCR was applied to confirm the m6A modification of METTL3 targets.ResultsMETTL3 was defective in the CD4+ T cells of SLE patients. METTL3 expression varied following CD4+ T-cell activation and effector T-cell differentiation in vitro. Pharmacological inhibition of METTL3 promoted the activation of CD4+ T cells and influenced the differentiation of effector T cells, predominantly Treg cells, in vivo. Moreover, METTL3 inhibition increased antibody production and aggravated the lupus-like phenotype in cGVHD mice. Further investigation revealed that catalytic inhibition of METTL3 reduced Foxp3 expression by enhancing Foxp3 mRNA decay in a m6A-dependent manner, hence suppressing Treg cell differentiation.ConclusionIn summary, our findings demonstrated that METTL3 was required for stabilizing Foxp3 mRNA via m6A modification to maintain the Treg differentiation program. METTL3 inhibition contributed to the pathogenesis of SLE by participating in the activation of CD4+ T cells and imbalance of effector T-cell differentiation, which could serve as a potential target for therapeutic intervention in SLE.

GOT1 regulates CD8+ effector and memory T cell generation

T cell activation, proliferation, function, and differentiation are tightly linked to proper metabolic reprogramming and regulation. By using [U-13C]glucose tracing, we reveal a critical role for GOT1 in promoting CD8+ Tcell effector differentiation and function. Mechanistically, GOT1 enhances proliferation by maintaining intracellular redox balance and serine-mediated purine nucleotide biosynthesis. Further, GOT1 promotes the glycolytic programming and cytotoxic function of cytotoxic T lymphocytes via posttranslational regulation of HIF protein, potentially by regulating the levels of α-ketoglutarate. Conversely, genetic deletion of GOT1 promotes the generation of memory CD8+ Tcells.

Possible immunological mechanisms in COVID-19 patients with immune thrombocytopenic purpura

Millions of people around the world were, or are still involved with COVID-19 due to infection with SARS-CoV-2. In addition to hallmark symptoms, thrombotic problems, lymphopenia, and thrombocytopenia have also been reported in COVID-19 patients, of which ITP is the most common and occurs in more than one-third of COVID-19 patients. Hyperinflammation, cytokine storms, and generally immune dysregulation in a percentage of patients develop the main consequences of diseases such as ALI, ARDS and multiple organ failure. Some of the important events in the immunopathogenesis of this disease are disruption of T-cell effector differentiation and the destructive role of Th17 lymphocytes, neutrophil function and inflammatory macrophages. NLRP3-inflammasome hyperactivity causes serious dysfunction of innate immune cells and, consequently, T lymphocytes in many inflammatory disorders, most notably in the COVID-19. A closer look at the immunopathogenesis of ITP and COVID-19 brings us to common ground. The purpose of this study was to review and summarize the findings of various studies on the immunopathogenesis of ITP and its possible causes in COVID-19. Finally, enhanced differentiation of Th17 and Th1, the cell death called as pyroptosis, hyperinflammation and dysfunction of inflammatory neutrophils and macrophages, and NLRP3- inflammasome hyperactivity are important factors in the development of thrombocytopenia in patients with COVID-19. Further studies are needed to better understand immunopathogenesis and effective treatments for ITP, especially in inflammatory disorders

A Sampling of Highlights from the Literature

CRISPR-based screening can enhance understanding of T-cell biology (from marius walter via Wikimedia Commons)Using CRISPR-based screening, two groups have identified canonical BRG1/BRM-associated factor (cBAF) complex components as regulators of T-cell differentiation and function that are important for antitumor immunity. Guo et al. find cBAF levels influence the fate of activated CD8+ T cells: high levels promote effector T-cell differentiation and low levels promote memory T-cell differentiation. Belk et al. show cBAF complex components, including Arid1a, promote T-cell acquisition of epigenetic features of exhaustion and limit T-cell persistence. Reducing cBAF activity, either pharmacologically (Guo et al.) or genetically (Belk et al.), improves tumor control in mouse models. The two studies suggest modulating the cBAF complex may improve T cell–based cancer immunotherapy.Guo A, …, Green DR. Nature 2022 June 22;607:135–141.Belk JA, …, Satpathy AT. Cancer Cell 2022 July 11;40:768–86.E7.S. epidermis specific T-cell responses can drive skin irAEs (from NIAID via NIH Flickr)Increasing our understanding of the mechanisms underlying immune-related adverse events (irAEs) is important for optimizing the use of immune checkpoint inhibitors (ICI). Using a mouse model of skin irAEs, Hu et al. find that exposure to Staphylococcus epidermis at the time of anti-CTLA4 treatment induces an inflammatory response resembling skin irAEs in patients treated with ICIs. The response is driven by S. epidermis–specific T cells that produce IL17 and leads to the emergence of S. epidermis–specific memory T cells. Further analysis will determine whether modulating cross-talk between the host immune system and microbiota can alleviate skin irAEs.Hu ZI, …, Belkaid Y. PNAS 2022 June 21;119:e2200348119.Phase II clinical trial results testing chemoimmuno-therapy in PDAC (via Rawpixel)Immunotherapy has so far been ineffective in the treatment of pancreatic ductal adenocarcinoma (PDAC). Padrón et al. report results from a phase II clinical trial evaluating chemotherapy combined with the anti–PD-1 nivolumab or the CD40-agonist sotigalimab, or with both together. The only group of patients among whom the 1-year overall survival rate was significantly greater than a historical control rate of 35% was the group assigned nivolumab/chemotherapy. Analysis of circulating and tumor biomarkers identified distinct immune signatures of improved outcome for patients receiving nivolumab/chemotherapy or sotigalimab/chemotherapy that could be used for patient selection in future clinical trials.Padrón LJ, …, Vonderheide RH. Nat Med 2022 June 3;28:1167–77.B cells can promote or suppress tumors depending on the TME (Carsten Tolkmit via Flickr)The role of B cells in the suppression or promotion of tumors remains unclear. In colorectal cancer (CRC), Wang et al. discover a specific regulatory B-cell subset expressing leucine-tRNA-synthase-2 (LARS2) and TGFβ1 that contributes to CRC progression by altering metabolic processes in the tumor microenvironment (TME). In a second study, Sagiv-Barfi et al. demonstrate that B-cell activation and antigen presentation are key to inducing antitumor T-cell responses, and both B and T cells are needed for efficacy of combination therapy with IL12, anti-OX40, and CpG in breast cancer. The two studies highlight how B-cell responses in the TME not only depend on cancer type, but also on B-cell phenotype and regulatory function.Wang Z, …, Chu Y. Immunity 2022 June 14;55:1067–81.E8.Sagiv-Barfi I, …, Levy R. Sci Immunol 2022 May 27;7:eabn5859.Profiling the diversity of tumor antigens (Andy Mitchell via Wikimedia Commons)A greater understanding of in vivo cancer-specific patterns of peptide presentation by MHC class I complexes is needed to improve the development of antigen-specific immunotherapies. Jaeger et al. develop and use a new genetically engineered mouse model to profile the cancer immunopeptidome from autochthonous pancreatic ductal adenocarcinoma and lung adenocarcinomas. The authors find little correlation between mRNA abundance and peptide detection, suggesting significant posttranslational regulation of tumor-specific peptide presentation. Dendritic cell vaccines loaded with lung adenocarcinoma peptides uniquely identified by the assay induce antigen-specific T-cell responses ex vivo, highlighting the model’s ability to capture the cancer immunopeptidome.Jaeger AM, …, Jacks T. Nature 2022 June 15;607:149–55.A new map of human immune cell development (tzunghaor via openclipart)Understanding how and where the immune system develops can provide a better understanding of cancer origin and facilitate the development of novel immunotherapies. Through single-cell profiling and spatial transcriptomics of nine prenatal tissues, Suo et al. have generated a comprehensive atlas of the developing human immune system. Highlights of the findings include the discovery that human immune development occurs across multiple tissue sites, including sites not considered primary hematopoietic organs; the identification of a population of prenatal B1 cells; and the demonstration that unconventional T cells are selected on thymocytes. This road map provides a resource for advancing immune-based science and medicine.Suo C, …, Teichmann SA. Science 2022 May 12;376:eabo0510.

T Cell-Intrinsic Vitamin A Metabolism and Its Signaling Are Targets for Memory T Cell-Based Cancer Immunotherapy.

Memory T cells play an essential role in infectious and tumor immunity. Vitamin A metabolites such as retinoic acid are immune modulators, but the role of vitamin A metabolism in memory T-cell differentiation is unclear. In this study, we identified retinol dehydrogenase 10 (Rdh10), which metabolizes vitamin A to retinal (RAL), as a key molecule for regulating T cell differentiation. T cell-specific Rdh10 deficiency enhanced memory T-cell formation through blocking RAL production in infection model. Epigenetic profiling revealed that retinoic acid receptor (RAR) signaling activated by vitamin A metabolites induced comprehensive epigenetic repression of memory T cell-associated genes, including TCF7, thereby promoting effector T-cell differentiation. Importantly, memory T cells generated by Rdh deficiency and blocking RAR signaling elicited potent anti-tumor responses in adoptive T-cell transfer setting. Thus, T cell differentiation is regulated by vitamin A metabolism and its signaling, which should be novel targets for memory T cell-based cancer immunotherapy.

Immunoregulatory effects of testosterone supplementation combined with exercise training in men with Inclusion Body Myositis: a double-blind, placebo-controlled, cross-over trial.

ObjectivesSporadic Inclusion Body Myositis (IBM) is an inflammatory muscle disease affecting individuals over the age of 45, leading to progressive muscle wasting, disability and loss of independence. Histologically, IBM is characterised by immune changes including myofibres expressing major histocompatibility complex molecules and invaded by CD8+ T cells and macrophages, and by degenerative changes including protein aggregates organised in inclusion bodies, rimmed vacuoles and mitochondrial abnormalities. There is currently no cure, and regular exercise is currently the only recognised treatment effective at limiting muscle weakening, atrophy and loss of function. Testosterone exerts anti‐inflammatory effects, inhibiting effector T‐cell differentiation and pro‐inflammatory cytokine production.MethodsWe conducted a double‐blind, placebo‐controlled, cross‐over trial in men with IBM, to assess whether a personalised progressive exercise training combined with application of testosterone, reduced the inflammatory immune response associated with this disease over and above exercise alone. To assess intervention efficacy, we immunophenotyped blood immune cells by flow cytometry, and measured serum cytokines and chemokines by Luminex immunoassay.ResultsTestosterone supplementation resulted in modest yet significant count reduction in the classical monocyte subset as well as eosinophils. Testosterone‐independent immunoregulatory effects attributed to exercise included altered proportions of some monocyte, T‐ and B‐cell subsets, and reduced IL‐12, IL‐17, TNF‐α, MIP‐1β and sICAM‐1 in spite of interindividual variability.ConclusionOverall, our findings indicate anti‐inflammatory effects of exercise training in IBM patients, whilst concomitant testosterone supplementation provides some additional changes. Further studies combining testosterone and exercise would be worthwhile in larger cohorts and longer testosterone administration periods.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

Nanotubes mediate mitochondrial transfer to tumor cells (by Michael Ströck via Wikimedia Commons)Tumors use various mechanisms to escape immune detection in the tumor microenvironment (TME). Saha et al. show that tumor cells form nanotubes that physically interact with immune cells in the breast cancer TME. Mitochondria are then transferred from immune cells to tumor cells, leading to tumor metabolic enhancement and loss of specific immune-cell subsets needed for antitumor responses. Targeting nanotube formation reversed these effects and when combined with immune checkpoint blockade, resulted in improved outcomes in a murine breast cancer model. The data highlight a new immune “hijacking” mechanism that tumors cells could use to promote cancer progression.Saha T, …, Sengupta S. Nat Nanotechnol 2021 Nov 18. DOI: 10.1038/s41565-021-01000-4.Lung cancers can become infiltrated by dysfunctional CD8+ T cells (by Eric Synder via NCI Visuals Online)Many non–small cell lung cancers (NSCLC) infiltrated with CD8+ T cells are resistant to immune checkpoint blockade. In an orthotopic NSCLC model, Horton et al. find lung tumors are infiltrated with CD8+ T cells but do not respond to combination anti–CTLA-4 and anti–PD-1. The CD8+ T cells have a dysfunctional phenotype molecularly distinct from T-cell exhaustion. The dysfunctional phenotype is induced during T-cell priming in lymph nodes and characterized by failure to acquire an effector program. Treatment with IL2 and IL12 restores effector T-cell differentiation. As CD8+ T cells from NSCLC patients have an analogous gene expression program of dysfunction, the data have translational potential.Horton BL, …, Spranger S. Sci Immunol 2021 Oct 29;6:eabi8800.γδ T cells infiltrate many tumor types and are highly cytotoxic (from Fig. 5C of Gherardin et al., Cancer Immunol Res 2021)Antibodies specific for butyrophilin 3A (BTN3A) can induce Vγ9Vδ2 T–cell cytolytic activity. De Gassart et al. have engineered a humanized BTN3A-specific antibody (ICT01) that induces Vγ9Vδ2 T–cell killing of several human tumor cell lines and primary tumor cells in vitro and xenografted tumors in mice following Vγ9Vδ2 T–cell transfer. Preliminary data from six patients with solid tumors administered ICT01 in the phase I/IIa EVICTION clinical trial (NCT04243499) show the antibody can be well tolerated, has caused no major safety events, and appears to induce broad immune-cell infiltration of tumors. Further studies should reveal the full potential of this strategy.De Gassart A, …, Frohna P. Sci Transl Med 2021 Oct 20;13:eabj0835.Histamine can suppress antitumor responses (by NEUROtiker via Wikimedia Commons)Identification of obstacles to effective antitumor immunity sometimes reveals surprising participants. Li et al. find that histamine, a mediator of allergic responses, plays an inhibitory role in antitumor responses. Histamine is frequently found in the tumor microenvironment and can activate macrophages via histamine receptor H1 (HRH1), promoting polarization to a suppressive M2-like phenotype and upregulation of the inhibitory checkpoint VISTA. Such macrophages suppress T-cell responses and promote tumor growth and therapy resistance. Knockout of HRH1 or use of antihistamines rescues experimental antitumor responses. The study highlights a role for histamine in modulating antitumor responses and suggests antihistamines as potential adjuvants for cancer immunotherapies.Li H, …, Yu D. Cancer Cell 2021 Nov 24. DOI: 10.1016/j.ccell.2021.11.002.PC-CAR T cells could unlock the immunotherapeutic potential of intracellular antigens (by Paris on Ponce & Le Maison Rouge via Flickr)Developing chimeric receptor (CAR) T cells specific for intracellular tumor-specific antigens is challenging because these antigens are only available to the immune system as peptides presented on the cell surface by MHC class I. Yarmarkovich et al. overcome this challenge by generating peptide-centric CARs (PC-CAR), which can recognize peptides derived from intracellular oncoproteins presented by multiple HLA types. PC-CAR T cells targeting a peptide from the neuroblastoma oncoprotein PHOX2B selectively kill neuroblastoma cells in vitro and clear tumors transplanted into mice. This study indicates PC-CARs have the potential to unlock the vast number of intracellular oncoproteins as potential targets for CAR T-cell immunotherapy.Yarmarkovich M, …, Maris JM. Nature 2021 Nov 3;599:477–84.TILs accumulate in ovarian cancer islets (from Fig. 1C of Heath et al., Cancer Immunol Res 2021)The tumor microenvironment (TME) factors that regulate exhaustion of CD8+ tumor-infiltrating lymphocytes (TIL) are not fully understood. Duraiswamy et al. found activated, tumor-specific CD8+ TILs concentrated in ovarian cancer islets, where they are closely associated with myeloid antigen-presenting cells (APC) but restrained by PD-1 signaling. Activated islet-resident APCs can provide CD28 costimulation, which is required for exhausted TILs to effectively respond to anti–PD-1 treatment in vitro. These findings suggest that a TME with insufficient CD28 signaling from myeloid APCs could explain poor responses to anti–PD-1 in tumors with substantial TIL populations and that activating the APCs with reagents such as anti-CD40 may rescue therapeutic responses.Duraiswamy J, …, Coukos G. Cancer Cell 2021 Dec 13;39:1623–42.e20.

Targeting Pim2 for Improving T-Cell Effector Function and Promoting Cancer Immunotherapy

The Provirus Integration sites for Moloney murine leukemia virus (Pim) kinases are a highly conserved family of serine/threonine kinases. The Pim kinase family is composed of three different isoforms, Pim1, Pim2, and Pim3, which have been studied extensively in tumorigenesis and as a potential therapeutic target in various cancers. We previously reported an unexpected role of Pim2 in negatively regulates T-cell responses to alloantigen and tumor (JCI, 2015, PMID: 29781812). However, the mechanisms by which Pim2 modulates T-cell responses remain largely undefined. In the current study, using genetic Pim2-deficient mouse, we demonstrated a key role of Pim2 in regulating T-cell hemostatic and anti-tumor responses in aging, hematopoietic cell transplantation (HCT), and antigen-specific adoptive T-cell therapy (ACT). We observed that Pim2 was critical for T cells to retain quiescent in aged mice, as thymic Treg development was impaired while effector T-cell differentiation in lymphoid organs, including Tc1/Th1, Tc17/Th17 and follicular helper T cells, was increased in Pim2-deficient mice, but not in Pim1/Pim3-deficient mice. Furthermore, Pim2-deficient mice were capable to completely eradicate syngeneic breast cancer (NT2.5) growth (Figure A). During antigen specific anti-tumor response, adoptively transferred Pim2 -/- CD8 T cells showed enhanced ability for controlling established NT2.5 breast cancer and B16 melanoma (Figure B, C). Mechanistically, loss of Pim2 promoted G1 to S phase cell-cycle progression while reduced apoptosis in CD8 T cells. Pim2 -/- CD8 T cells exhibited elevated effector cytokine production while maintained higher levels of CD62L expression, leading to superior effector function, persistence and anti-tumor activity. Reduced differentiation of exhausted and suppressive subsets were observed in Pim2 -/- CD8 T cells after being adoptively transferred in tumor-bearing mice. In addition, Pim2 deficiency was associated with a higher metabolic potential, reflected by increased glycolysis and oxidative phosphorylation, which was at least partially attributed to a decreased level of autophagy in Pim2 -/- CD8 T cells. To further evaluate the clinical translation potential, we applied a Pim2-specific inhibitor (JP11646) and found that blocking Pim2 improved graft-versus-leukemia activity after autologous HCT and also enhanced CD8 T-cell mediated anti-melanoma effects after ACT in mice (Figure B, C). Furthermore, blocking Pim2 using JP11646 promoted human CD8 T-cell response during polyclonal stimulation and enhanced expansion, effector function and tumor killing ability of human melanoma antigen-specific CD8 T cells (data not shown) and CD19 CAR-T cells (Figure D). Our work demonstrated that Pim2 is a potent and distinct regulator of differentiation and maintenance of T effector cells through modulating metabolism and autophagy. Specifically target Pim2 can serve as a novel strategy for improving cancer immunotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 Tcell effector differentiation.

SUMMARYTo gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.

Vitamin D deficiency after allogeneic hematopoietic cell transplantation promotes T-cell activation and is inversely associated with an EZH2-ID3 signature

Vitamin D promotes a shift from a proinflammatory to a more tolerogenic immune state in allogeneic hematopoietic cell transplant (HCT) recipients. The dominant mechanism responsible for this shift has not been elucidated. We took a multifaceted approach to evaluating the clinical and immunologic impact of low vitamin D levels in 53 HCT recipients. We used 28-plex flow cytometry for immunophenotyping, serum cytokine levels, T-cell cytokine production, and T-cell whole genome transcription. The median day-30 vitamin D level was 20 ng/mL, and deficiency was common in younger patients undergoing myeloablative transplantation. Low vitamin D levels were associated with a high CD8/Treg ratio, increased serum levels and T-cell production of proinflammatory cytokines, and a gene expression signature of unrestrained T-cell proliferation and epigenetic modulation through the PRC2/EZH2 complex. Immunophenotyping confirmed a strong association between high levels of vitamin D and an activated EZH2 signature, characterized by overexpression of ID3, which has a role in effector T-cell differentiation. Our findings demonstrate the critical role of vitamin D in modulating T-cell function in human GVHD and identify a previously undescribed interaction with EZH2 and ID3, which may impact effector differentiation and has implications to cell therapies and other forms of cancer immunotherapy.© 20XX American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Single-cell immunopathology of systemic contact allergy associated with corticosteroids

Rationale Allergic contact dermatitis (ACD) is a classic delayed hypersensitivity reaction commonly associated with corticosteroids that can rarely manifest as a delayed rash following systemic administration. Methods A 50-year old woman developed local edema and delayed spreading rash following interarticular injection with dexamethasone and methylprednisolone acetate (MA). 6-months following this intradermal skin testing (IDST) was positive to MA and methylprednisolone sodium succinate, and IDST and patch test negative to other corticosteroids. She tolerated dexamethasone challenge. Cells were isolated from 4-mm punch biopsies from 48-hour positive IDST and unaffected skin with liberase digestion and sorted on CD3. scTCR and scRNA-seq were performed using plate-based assays (Smart-seq-2). Data was filtered using Seurat and analysed with Visual Genomics Analysis Studio (VGAS) software. Results Histopathology showed a superficial perivascular dermatitis with epidermal spongiosis in keeping with ACD. T-cells were 18x more prevalent in affected skin and those from both affected and unaffected sites were predominantly CD8+ T-cells that expressed CD45RO and polyclonal TCR alpha beta. Differentially expressed genes (DEG) in T-cells from affected skin reflected homing (CCR7,S1PR1), T-cell activation and proliferation (TNFRS9, GRAP2, ZYG11A, ZHX1), T-cell effector differentiation (IL-21R), and stress survival (EEF1A1, IFI6) without representation of markers of T-cell residency (ITGAE) or regulation. Conclusions We demonstrate insights into the immunopathogenesis of drug-induced ACD by showing that IDST+ methylprednisolone associated ACD is corticosteroid-specific and associated with a polyclonal population of primarily CD45RO+ memory CD8+ T-cells showing upregulation of markers of homing, T-cell activation, proliferation and differentiation but lacking markers of T-cell residency and regulation.

Haematopoietic PI3-kinase delta deficiency profoundly impairs regulatory T-cell biology and thereby protection against atherosclerosis

Abstract Introduction and purpose Atherosclerosis is a chronic inflammatory disease of arteries, critically involving leukocytes like macrophages as well as T and B lymphocytes. Macrophages are major drivers of disease through the ingestion of lipoproteins, foam cell formation, and secretion of inflammatory mediators. Although macrophages outnumber other leukocytes in atherosclerotic plaques, T and B cells can shape the course of disease by promoting or mitigating inflammatory responses. Leukocytes highly express the phosphoinositide 3-kinase isoform delta (PI3Kd), exerting a key role in the regulation of immune responses including activation, proliferation, differentiation, and effector functions. Therefore, PI3Kd represents a promising target for the modulation of inflammatory diseases. Consequently, we aimed to analyse the role of PI3Kd in leukocytes during atherogenesis. Methods and results To investigate the role of PI3Kd in atherosclerosis, bone marrow from PI3Kd−/− or PI3Kd+/+ mice was transplanted into LDLR−/− mice. After a 6-weeks-challenge by high fat diet, PI3Kd−/− recipient LDLR−/− mice displayed profoundly impaired CD4+ and CD8+ T-cell numbers, CD4+ T-cell activation, CD4+ effector T-cell differentiation, and proatherogenic CD4+ T-helper (Th) 1 responses in para-aortic lymph nodes and spleen compared with PI3Kd+/+ transplanted controls. Surprisingly, the net effect of PI3Kd deficiency was a substantial increase of aortic inflammation and atherosclerosis in LDLR−/− mice. Whereas plaque content and functions of macrophages including foam cell formation, efferocytosis, and cytokine secretion remained unaffected, haematopoietic PI3Kd ablation strongly reduced mature B cells and serum immunoglobulins in LDLR−/− mice. Importantly, PI3Kd deficiency severely impaired numbers and immunosuppressive functions of regulatory CD4+ T cells (Tregs) in spleen, para-aortic lymph nodes, and plaques of LDLR−/− mice. Consequently, adoptive transfer of PI3Kd+/+ Tregs fully constrained the plaque burden in PI3Kd−/− transplanted LDLR−/− mice without affecting B-cell numbers and serum immunoglobulins, whereas adoptively transferred PI3Kd−/− Tregs were unable to relieve atherosclerosis progression. Conclusions Here, we demonstrate that PI3Kd plays a crucial role in Tregs, Th1 cells, and B cells during atherogenesis. Lack of PI3Kd signalling specifically in atheroprotective Treg responses outplays its impact on proatherogenic Th1 and B-cell responses, thus leading to aggravated atherosclerosis. Hence, PI3Kd is a key regulator of Treg biology and thereby protects against atherosclerosis. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Marga and Walter Boll Foundation

Abstract IA14: mTOR as a central regulator of T-cell activation, differentiation, and function

Abstract While the exquisite specificity of the T-cell receptor (TCR) MHC + peptide interaction heralds recognition, it is ultimately the cues from the immune microenvironment that dictate the outcome of this recognition. Our lab has proposed that mTOR plays a critical role in integrating signals and thus determining the outcome of antigen recognition. Indeed, we have demonstrated that mTORC1 and mTORC2 signaling can selectively guide CD4+ effector and regulatory T-cell differentiation and function as well as CD8+ effector and memory T-cell differentiation and function. Recent studies have revealed that mTOR performs this function in part by coordinating T-cell activation and function with metabolic reprogramming. Consequently, targeting downstream mTOR signaling and metabolism can promote T-cell effector function and persistence in order to enhance cancer immunotherapy. Likewise, targeting mTOR can inhibit T-effector function and promote the generation of regulatory T cells to treat autoimmune disease and prevent transplantation rejection. Citation Format: Jonathan Powell, Chirag Patel, Hong Sun, Michael Claiborne. mTOR as a central regulator of T-cell activation, differentiation, and function [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA14.

Abstract 1044: Frequency of circulating CX3CR1+ CD8+ T cells to predict response to immune checkpoint inhibitor therapy

Abstract While immune checkpoint inhibitors (ICI) have revolutionized treatment for various cancers including non-small cell lung cancer (NSCLC), only a subset of patients receives durable clinical benefit. Discovery of blood-based biomarkers that reflect dynamic change of tumor microenvironment, and predict response to ICI will markedly improve current treatment regimens. Emerging evidence has shown that the frequency of peripheral blood (PB) CD8+ T cells expressing CX3CR1, a marker of effector T-cell differentiation increases in patients receiving ICI. However, no studies have rigorously evaluated the utility of CX3CR1 as a PB biomarker to predict response to ICI, and identify responders and non-responders. Here, we evaluated the utility of circulating T-cell biomarkers to predict response to ICI therapy in preclinical models and NSCLC patients (n=35). We found successful treatment with ICI significantly increases the frequency of PB CX3CR1+ CD8+ T-cell subsets that enrich neoantigen- as well as shared tumor-associated antigen-specific T cells in mice bearing MC38 and CT26 colon adenocarcinoma. Significantly increased expression of IFN-γ and Ki67 suggests that these subsets are highly proliferative effector CD8+ T cells. Interestingly, upregulation of Ki67 was transient; however, expression of CX3CR1 remained high on tumor-specific tetramer+ CD8+ T cells. Moreover, high-throughput sequencing of the T-cell receptor (TCR) identified significantly higher clonality and overlapping TCR repertoires in between peripheral CX3CR1+ CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs), suggesting the potential utility as a dynamic biomarker early on-treatment. Most importantly, changes in the frequency of PB CX3CR1+ CD8+ T cells associate with response to ICI therapy in individual mice. Phenotypic analysis of PB CD8+ T cells from 35 NSCLC patients undergoing PD-1/PD-L1 blockade revealed that the maximum percent change of these T-cell subsets from baseline identified responders and non-responders as early as 3 weeks from the initiation of ICI therapy with higher sensitivity, specificity, positive and negative predictive value compared to PD-L1 expression in the tumor, tumor mutational burden, and pre-existing TILs. Importantly, TCR sequencing confirmed clonally-expanding TCR repertoires within the T-cell subsets that were also found in TILs. Collectively, the frequency of circulating CX3CR1+ CD8+ T cells correlates with response to ICI therapy, and identifies responders vs non-responders early on-treatment. These findings provide a solid foundation for future development of clinical trials with large cohorts of patients undergoing ICI therapy not only for NSCLC, but also for a wide variety of malignancies. Citation Format: Takayoshi Yamauchi, Toshifumi Hoki, Takaaki Oba, Kristopher Attwood, Sebastiano Battaglia, Igor Puzanov, Hongbin Chen, Grace Dy, Brahm Segal, Marc Ernstoff, Fumito Ito. Frequency of circulating CX3CR1+ CD8+ T cells to predict response to immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1044.

CD96 functions as a co-stimulatory receptor to enhance CD8+ Tcell activation and effector responses.

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes Tcell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate Tcell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ Tcells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ Tcells. Crosslinking of CD96 on human or mouse CD8+ Tcells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ Tcells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector Tcell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ Tcells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ Tcell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ Tcell activation and effector function.

The Multiparametric Analysis of Mitochondrial Dynamics in T Cells from Cryopreserved Peripheral Blood Mononuclear Cells (PBMCs).

The analysis of mitochondrial dynamics within immune cells allows us to understand how fundamental metabolism influences immune cell functions, and how dysregulated immunometabolic processes impact biology and disease pathogenesis. For example, during infections, mitochondrial fission and fusion coincide with effector and memory T-cell differentiation, respectively, resulting in metabolic reprogramming. As frozen cells are generally not optimal for immunometabolic analyses, and given the logistic difficulties of analysis on cells within a few hours of blood collection, we have optimized and validated a simple cryopreservation protocol for peripheral blood mononuclear cells, yielding >95% cellular viability, as well as preserved metabolic and immunologic properties. Combining fluorescent dyes with cell surface antibodies, we demonstrate how to analyze mitochondrial density, membrane potential, and reactive oxygen species production in CD4 and CD8 T cells from cryopreserved clinical samples.

TGFβ Programs Central Memory Differentiation in Ex Vivo-Stimulated Human T Cells.

The adoptive transfer of ex vivo-expanded T cells is a promising approach to treat several malignancies. Several lines of evidence support that the infusion of T cells with early memory features, capable of expanding and persisting after transfer, are associated with better outcomes. We report herein that exposure to exogenous TGFβ during human T-cell stimulation ex vivo leads to the accumulation of early/central memory (Tcm) cells. Exposure to TGFβ suppressed the expression of BLIMP-1, a key orchestrator of effector T-cell differentiation, and led to the upregulation of the memory-associated transcription factor ID3. Accordingly, this was associated with an early memory transcriptional signature in both CD4+ and CD8+ T-cell subsets. The T cells stimulated in the presence of TGFβ expanded normally, and displayed polyfunctional features and no suppressive activity. The adoptive transfer of ex vivo-stimulated T cells into immunodeficient mice confirmed that TGFβ-conditioned cells had an enhanced capacity to persist and mediate xenogeneic graft-versus-host disease, as predicted by their early T-cell memory phenotype. Chimeric antigen receptor-expressing T cells generated in the presence of exogenous TGFβ were cytotoxic and more effective at controlling tumor growth in immunodeficient animals. This work unveils a new role for TGFβ in memory T-cell differentiation and indicates that TGFβ signaling may be harnessed to program Tcm differentiation in the context of ex vivo T-cell stimulation for adoptive immunotherapy in humans.

Phase 1 Open-Label, Multicenter Study of First-in-Class RORγ Agonist LYC-55716 (Cintirorgon): Safety, Tolerability, and Preliminary Evidence of Antitumor Activity

Transcription factor retinoic acid receptor-related orphan receptor γ (RORγ) regulates type 17 effector T-cell differentiation and function and is key to immune cell regulation. Synthetic RORγ agonists modulate immune cell gene expression to increase effector T-cell activity and decrease immune suppression. A phase 1 study evaluated the safety and tolerability of LYC-55716 (cintirorgon), a first-in-class, oral, small-molecule RORγ agonist in adults with relapsed/refractory metastatic cancer. Patients received 28-day treatment cycles of oral LYC-55716; dose and dosing regimen were determined according to pharmacokinetic profile and safety. Primary endpoints were safety and tolerability. Secondary endpoints included pharmacokinetics and objective tumor response rate. No dose-limiting toxicities occurred among the 32 enrolled patients who received LYC-55716 150 mg BID to 450 mg BID. Treatment-related adverse events (AE) were primarily grade 1-2 and included diarrhea (n = 11), fatigue (n = 7), anemia (n = 4), decreased appetite (n = 4), and nausea (n = 4). Grade 3 AEs were anemia (n = 2), elevated gamma-glutamyl transferase (n = 1), and hypophosphatemia (n = 1). Pharmacokinetic concentrations achieved levels expected for target gene regulation. Pharmacodynamic results indicated RORγ pathway engagement. Two patients (NSCLC and sarcomatoid breast cancer) had confirmed partial responses; 11 had disease stabilization for 2 to 12 months (6 received >4 months of treatment). These data support the safety and tolerability of LYC-55716 and selection of 450 mg BID dose for a phase 2a study assessing LYC-55716 clinical activity, safety, and biomarkers in patients with NSCLC, head and neck, gastroesophageal, renal cell, urothelial, and ovarian cancers.

Highlights of This Issue

Neuroendocrine tumors (NET) are rare neoplasms arising from diffusive neuroendocrine cells of various organs. Current systemic treatments for advanced or metastatic NET are limited. The clinical effect of VEGFR inhibitors has not been well characterized in extrapancreatic NET. In this phase II study by Xu and colleagues, surufatinib, a novel and selective VEGFR inhibitor, showed potent and durable antitumor activity in patients with well-differentiated advanced NET irrespective of the primary tumor origins; similar antitumor activity was observed in both VEGFR-inhibitor-naïve and pretreated patients. This study provided clinical evidence that surufatinib might be a promising therapeutic candidate for patients with well-differentiated advanced NET.95% of all peptide vaccines focus on cellular T cell immune responses. Bekaii-Saab and colleagues report a new paradigm in immunotherapy that focuses on humoral responses based on chimeric conformational B-cell epitope vaccines. In the phase 1b trial, the combination of two HER-2 peptide B-cell epitope vaccines engineered to represent the trastuzumab and pertuzumab binding sites was safe, exhibited antitumor activity, and showed preliminary indications that peptide vaccination may avoid therapeutic resistance and offer a promising alternative to monoclonal antibody therapies.The retinoic acid-related orphan receptor gamma (RORgamma) is a nuclear receptor transcription factor that acts as an immune cell master control switch through regulation of type 17 effector T-cell differentiation and function. In this phase 1 study, Mahalingam and colleagues evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of LYC-55716, a first in class, oral, selective RORgamma agonist. The study established a recommended dose for further study and provided evidence of clinical activity of LYC-55716. Based on the findings of this study, RORgamma agonists may represent a new approach to enhance antitumor immunity for patients with advanced cancers.Immunotherapy is used to treat several types of squamous cell carcinomas (SCC), but response rates are relatively low. In this study, Li and colleagues explored the immune landscape of esophageal, head and neck, lung, and cervical SCC. They identified and validated six immune subtypes, each of which was associated with distinct gene expression profiles, genetic aberrations, tumor-infiltrating immune cell composition and functional orientation, cytokine profiles, and clinical outcomes. This study provides a conceptual framework to understand the complex immune microenvironment of SCC and may have implications for the design of combination treatment strategies and selection of patients for immunotherapy.