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Patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) treated with immune checkpoint inhibitors (ICIs) are at risk of pneumonitis as well as pneumonia (combined henceforth as ICI-related pulmonary complications). Little is known about the cellular and molecular mechanisms underlying ICI-related pulmonary complications. We characterized lymphocytes from bronchoalveolar lavage (BAL) fluid and peripheral blood from seven AML/MDS patients with pulmonary symptoms after ICI-based therapy (ICI group) and four ICI-naïve AML/MDS patients with extracellular bacterial or fungal pneumonias (controls). BAL T cells in the ICI group were clonally expanded, and BAL IFNγ + IL-17 - CD8 + T and CXCR3 + CCR6 + Th17/Th1 cells were enriched in the ICI group. Our data suggest that these cells may play a critical role in the pathophysiology of ICI-related pulmonary complications. Understanding of these cell populations may also provide predictive and diagnostic biomarkers of ICI-related pulmonary complications, eventually enabling differentiation of pneumonitis from pneumonia in AML/MDS patients receiving ICI-based therapies., Competing Interests: SN has received research support from Kite/Gilead, Cellectis, Poseida, Merck, Acerta, Karus, BMS, Unum Therapeutics, Allogene, and Precision Biosciences; served as consultant and advisory board member for Kite/Gilead, Celgene, Novartis, Unum Therapeutics, Pfizer, Merck, Precision Biosciences, Cell Medica, Incyte, Allogene, Calibr, and Legend Biotech; and has patents related to cell therapy. ND has received research funding from Daiichi Sankyo, Bristol-Myers Squibb, Pfizer, Karyopharm, Sevier, Genentech, and ImmunoGen and has served in a consulting or advisory role for Daiichi Sankyo, Bristol-Myers Squibb, Pfizer, Novartis, Celgene, AbbVie, and Agios. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kim, Sheshadri, Shannon, Kontoyiannis, Kantarjian, Garcia-Manero, Ravandi, Im, Boddu, Bashoura, Balachandran, Evans, Faiz, Ruiz Vazquez, Divenko, Mathur, Tippen, Gumbs, Neelapu, Naing, Wang, Diab, Futreal, Nurieva and Daver.)

T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment 1 . The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction 2 . However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8 + T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

T cells are the master regulators of adaptive immune responses and maintenance of their tolerance is critical to prevent autoimmunity. However, in the case of carcinogenesis, the tumor microenvironment aids T-cell tolerance, which contributes to uncontrolled tumor growth. Recently, there has been significant progress in understanding the intrinsic extracellular (positive and negative costimulatory molecules on APCs) and intracellular mechanisms (E3 ubiquitin ligases, transcriptional and epigenetic repressors), as well as extrinsic mechanisms (Tregs and tolerogenic dendritic cells) that are required for the implementation and maintenance of T-cell tolerance. Ultimately, understanding and manipulating T-cell tolerance will help to break the tolerance state in cancer.

MAPKs are evolutionarily conserved immune regulators. MAPK phosphatases (MKPs) that negatively regulate MAPK activities have recently emerged as critical players in both innate and adaptive immune responses. MKP-1, also known as DUSP1, was previously shown to negatively regulate innate immunity by inhibiting pro-inflammatory cytokine production. Here, we found that MKP-1 is necessary in T cell activation and function. MKP-1 deficiency in T cells impaired the activation, proliferation, and function of T cells in vitro, associated with enhanced activation of JNK and reduced NFATc1 translocation into the nucleus. Consistently, MKP-1(-/-) mice were defective in anti-influenza immunity in vivo and resistant to experimental autoimmune encephalomyelitis. Our results thus demonstrate that MKP-1 is a critical positive regulator of T cell activation and function and may be targeted in treatment of autoimmune diseases.

Summary: In addition to signals from the T-cell receptor complex, it has been recognized for many years that a 'second' signal, most notably from CD28, is also important in T-cell activation. In the recent years, many new members of CD28 family as well as the molecules that share structural homology to CD28 ligands CD80 and CD86 have been discovered. Interestingly, some of these proteins function to dampen T-cell activation and regulate the induction of T-cell tolerance. Therefore, positive and negative costimulation are the two sides of the coin to fine tune T-cell receptor signaling to determine the outcome of T-cell receptor engagement-tolerance versus function.

T cell activation and tolerance are delicately regulated by costimulatory molecules. Although B and T lymphocyte attenuator (BTLA) has been shown as a negative regulator for T cell activation, its role in peripheral T cell tolerance induction in vivo has not been addressed. In this study, we generated a novel strain of BTLA-deficient mice and used three different models to characterize the function of BTLA in controlling T cell tolerance. In an oral tolerance model, BTLA-deficient mice were found resistant to the induction of T cell tolerance to an oral Ag. Moreover, compared with wild-type OT-II cells, BTLA(-/-) OT-II cells were less susceptible to tolerance induction by a high-dose OVA peptide administered i.v. Finally, BTLA(-/-) OT-I cells caused autoimmune diabetes in RIP-mOVA recipient mice. Our results thus demonstrate an important role for BTLA in the induction of peripheral tolerance of both CD4(+) and CD8(+) T cells in vivo.

Although interleukin (IL) 17 has been extensively characterized, the function of IL-17F, which has an expression pattern regulated similarly to IL-17, is poorly understood. We show that like IL-17, IL-17F regulates proinflammatory gene expression in vitro, and this requires IL-17 receptor A, tumor necrosis factor receptor-associated factor 6, and Act1. In vivo, overexpression of IL-17F in lung epithelium led to infiltration of lymphocytes and macrophages and mucus hyperplasia, similar to observations made in IL-17 transgenic mice. To further understand the function of IL-17F, we generated and analyzed mice deficient in IL-17F or IL-17. IL-17, but not IL-17F, was required for the initiation of experimental autoimmune encephalomyelitis. Mice deficient in IL-17F, but not IL-17, had defective airway neutrophilia in response to allergen challenge. Moreover, in an asthma model, although IL-17 deficiency reduced T helper type 2 responses, IL-17F-deficient mice displayed enhanced type 2 cytokine production and eosinophil function. In addition, IL-17F deficiency resulted in reduced colitis caused by dextran sulfate sodium, whereas IL-17 knockout mice developed more severe disease. Our results thus demonstrate that IL-17F is an important regulator of inflammatory responses that seems to function differently than IL-17 in immune responses and diseases.

T cell activation and differentiation is accompanied and mediated by transcriptional reprogramming. The NFATc1 transcription factor is strongly induced upon T cell activation and controls numerous genes involved in the T cell effector function. However, its regulation by physiological stimuli in primary T cells has not been well understood. We previously found that ICOS synergizes with TCR and CD28 to greatly enhance NFATc1 expression in primary T cells. In this study, we have examined the signaling mechanisms whereby costimulation regulates NFATc1 expression. We found that CD28 and ICOS regulate sustained PI3K activity in primary T cells, which is required for NFATc1 up-regulation. CD28 and ICOS costimulation, possibly through Itk, a Tec kinase downstream of the PI3K, enhanced phosphorylation of phospholipase C gamma1 and increased and sustained Ca(2+) flux in T cells. Costimulation of T cells potentiated transcription of the Nfatc1 gene P1 promoter in a PI3K-dependent manner. This work demonstrates an important role for costimulatory receptors in sustaining T cell activation programs leading to Nfatc1 gene transcription and has implications in our understanding of the immune response and tolerance.

Activated in immune responses, T lymphocytes differentiate into effector cells with potent immune function. CD28 is the most prominent costimulatory receptor for T-cell activation. However, absence of CD28 costimulation did not completely impair effector function of CD4 or CD8 T cells. Moreover, increasing number of costimulatory molecules are recently found on antigen-presenting cells to regulate T-cell activation. To understand the molecular mechanisms that determine T-cell function or tolerance, we have collectively examined the roles of positive and negative costimulatory molecules. Antigen-specific naïve CD4 and CD8 T cells, only when activated in the absence of both CD28 and ICOS pathways, were completely impaired in effector function. These tolerant T cells not only were anergic with profound defects in TcR signal transduction but also completely lacked expression of effector-specific transcription factors. T-cell tolerance induction in this system requires the action by negative costimulatory molecules; T-cell proliferation and function was partially restored by inhibiting PD-1, B7-H3 or B7S1. This work demonstrates that T-cell function or tolerance is controlled by costimulatory signals.

Proper T cell activation and function are regulated by the innate immune system, importantly through positive and negative costimulatory molecules of the B7 superfamily. Inducible costimulator (ICOS) is expressed on T cells after T cell activation. A ligand for ICOS, B7h (also known as B7RP-1), is expressed on B cells and induced in nonlymphoid tissues by TNF-alpha. The wide distribution of B7h may play essential roles in different phases and types of immune function. In this review, we summarize the latest data about inducible costimulator and its receptor and their roles in regulation of immune and autoimmune responses.

T helper (Th) cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a recently identified costimulatory receptor expressed on activated T cells. A ligand for ICOS, B7h, is expressed on B cells and other types of antigen-presenting cells (APC). Although ICOS has been shown to be essential in T cell activation and differentiation, the regulatory roles of B7h at different stages of T cell immune responses have not been examined genetically. In this study, we generated and analyzed B7h-deficient mice. We present evidence that B7h is the only ligand for ICOS, and ICOS, its only corresponding receptor. Th cells, when activated with B7h-deficient APC, exhibited reduced proliferation and IL-2 production. In addition, Th cells produced significantly reduced amounts of IL-4 and -13 after differentiation at the presence of B7h-/- APC. This cytokine defect was associated with a deficiency in c-Maf expression and could be rescued completely by c-Maf overexpression in T cells. Furthermore, we showed that effector T cells, when restimulated in the presence of B7h-deficient APC, exhibited reduced Th2 cytokine production. Therefore, B7h is required for proper Th cell activation, differentiation, and effector cytokine expression.