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Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT), but how this affects immune responses broadly remains unknown. Using a preclinical model of CMV reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells, resulting in sustained IFN-[gamma] secretion, which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for IgG recycling, and rapid IgG loss. T cell-specific deletion of IL-6R led to persistence of recipient-derived, CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN-[gamma] in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invoked IFN-[gamma]-dependent EC injury and consequent IgG loss, leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury, thereby preserving humoral immunity after immunotherapy., Introduction The influence of inflammation on various aspects of immune responses is recognized, yet the extent of these effects remains largely unknown. This is especially true within clinical contexts in [...]

BACKGROUND. Precise stratification of patients with non-small cell lung cancer (NSCLC) is needed for appropriate application of PD-1/PD-L1 blockade therapy. METHODS. We measured soluble forms of the immune-checkpoint molecules PD-L1, PD-1, and CTLA-4 in plasma of patients with advanced NSCLC before PD-1/PD-L1 blockade. A prospective biomarker-finding trial (cohort A) included 50 previously treated patients who received nivolumab. A retrospective observational study was performed for patients treated with any PD-1/PD-L1 blockade therapy (cohorts B and C), cytotoxic chemotherapy (cohort D), or targeted therapy (cohort E). Plasma samples from all patients were assayed for soluble immune-checkpoint molecules with a highly sensitive chemiluminescence-based assay. RESULTS. Nonresponsiveness to PD-1/PD-L1 blockade therapy was associated with higher concentrations of these soluble immune factors among patients with immune-reactive (hot) tumors. Such an association was not apparent for patients treated with cytotoxic chemotherapy or targeted therapy. Integrative analysis of tumor size, PD-L1 expression in tumor tissue (tPD-L1), and gene expression in tumor tissue and peripheral [CD8.sup.+] T cells revealed that high concentrations of the 3 soluble immune factors were associated with hyper or terminal exhaustion of antitumor immunity. The combination of soluble PD-L1 (sPD-L1) and sCTLA-4 efficiently discriminated responsiveness to PD-1/PD-L1 blockade among patients with immune-reactive tumors. CONCLUSION. Combinations of soluble immune factors might be able to identify patients unlikely to respond to PD- 1/PD-L1 blockade as a result of terminal exhaustion of antitumor immunity. Our data suggest that such a combination better predicts, along with tPD-L1, for the response of patients with NSCLC. TRIAL REGISTRATION. UMIN000019674. FUNDING. This study was funded by Ono Pharmaceutical Co. Ltd. and Sysmex Corporation., Introduction Targeting of the immune system has provided clinical benefit for individuals with advanced solid tumors, including non-small cell lung cancer (NSCLC) (1). Currently approved immune-checkpoint inhibitors (ICIs) include monoclonal [...]

Corticosteroid treatment (CST) failure is associated with poor outcomes for patients with gastrointestinal (GI) graft-versus-host disease (GVHD). CST is intended to target the immune system, but the glucocorticoid receptor (GR) is widely expressed, including within the intestines, where its effects are poorly understood. Here, we report that corticosteroids (CS) directly targeted intestinal epithelium, potentially worsening immune-mediated GI damage. CS administered to mice in vivo and intestinal organoid cultures ex vivo reduced epithelial proliferation. Following irradiation, immediate CST mitigated GI damage but delayed treatment attenuated regeneration and exacerbated damage. In a murine steroid-refractory (SR) GVHD model, CST impaired epithelial regeneration, worsened crypt loss, and reduced intestinal stem cell (ISC) frequencies. CST also exacerbated immune-mediated damage in organoid cultures with SR, GR-deficient T cells or IFN-[gamma]. These findings correlated with CS-dependent changes in apoptosis-related gene expression and STAT3-related epithelial proliferation. Conversely, IL-22 administration enhanced STAT3 activity and overcame CS-mediated attenuation of regeneration, reducing crypt loss and promoting ISC expansion in steroid-treated mice with GVHD. Therefore, CST has the potential to exacerbate GI damage if it fails to control the damage-inducing immune response, but this risk may be countered by strategies augmenting epithelial regeneration, thus providing a rationale for clinical approaches combining such tissue- targeted therapies with immunosuppression., Introduction The epithelial lining of the gastrointestinal (GI) tract undergoes turnover every 5-7 days (1). This renewal is maintained by the cycling of leucine-rich, repeat-containing GPCR 5-positive ([Lgr5.sup.+]) intestinal stem [...]

Radiotherapy (RT) is considered immunogenic, but clinical data demonstrating RT-induced T cell priming are scarce. Here, we show in a mouse tumor model representative of human lymphocyte-depleted cancer that RT enhanced spontaneous priming of thymus-derived ([FOXP3.sup.+][Helios.sup.+]) Tregs by the tumor. These Tregs acquired an effector phenotype, populated the tumor, and impeded tumor control by a simultaneous, RT-induced [CD8.sup.+] cytotoxic T cell (CTL) response. Combination of RT with CTLA-4 or PD-1 blockade, which enables CD28 costimulation, further increased this Treg response and failed to improve tumor control. We discovered that upon RT, the CD28 ligands CD86 and CD80 differentially affected the Treg response. CD86, but not CD80, blockade prevented the effector Treg response, enriched the tumor-draining lymph node migratory conventional DCs that were positive for PD-L1 and CD80 ([PD-L1.sup.+][CD80.sup.+]), and promoted CTL priming. Blockade of CD86 alone or in combination with PD-1 enhanced intratumoral CTL accumulation, and the combination significantly increased RT-induced tumor regression and OS. We advise that combining RT with PD-1 and/or CTLA-4 blockade may be counterproductive in lymphocyte-depleted cancers, since these interventions drive Treg responses in this context. However, combining RT with CD86 blockade may promote the control of such tumors by enabling a CTL response., Introduction Immunotherapy by antibody-based immune checkpoint blockade (ICB) is a new treatment modality for multiple cancer types. However, only a minority of patients experience durable clinical responses (1), partly due [...]

Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in Union for International Cancer Control (UICC) stage IV CRC patients. Insights into differential site-specific reconstitution of tumor cells and the corresponding tumor microenvironment are still missing. Here, we analyzed the transcriptome of single cells derived from murine multivisceral CRC and delineated the intermetastatic cellular heterogeneity regarding tumor epithelium, stroma, and immune cells. Interestingly, we found an intercellular site-specific network of cancer- associated fibroblasts and tumor epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell-dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and--based on the results of our analysis--provided an auspicious site-specific immunomodulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition., Introduction Colorectal cancer (CRC) is the second most diagnosed cancer in females and the third most common in males, with about 0.8 million deaths worldwide per year (1). Whereas primary [...]

Background: The increasing prevalence of food sensitivities has been attributed to changes in gut microenvironment; however, ubiquitous environmental triggers such as inorganic nanoparticles (NPs) used as food additives have not been thoroughly investigated. Objectives: We explored the impact of the NP-structured food-grade silicon dioxide (fg-Si[O.sub.2]) on intestinal immune response involved in oral tolerance (OT) induction and evaluated the consequences of oral chronic exposure to this food-additive using a mouse model of OT to ovalbumin (OVA) and on gluten immunopathology in mice expressing the celiac disease risk gene, HLA-DQ8. Methods: Viability, proliferation, and cytokine production of mesenteric lymph node (MLN) cells were evaluated after exposure to fg-Si[O.sub.2]. C57BL/6J mice and a mouse model of OT to OVA were orally exposed to fg- Si[O.sub.2] or vehicle for 60 d. Fecal lipocalin-2 (Lcn-2), anti-OVA IgG, cytokine production, and immune cell populations were analyzed. Nonobese diabetic (NOD) mice expressing HLA-DQ8 (NOD/DQ8), exposed to fg-Si[O.sub.2] or vehicle, were immunized with gluten and immunopathology was investigated. Results: MLN cells exposed to fg-Si[O.sub.2] presented less proliferative T cells and lower secretion of interleukin 10 (IL-10) and transforming growth factor beta (TGF-[beta]) by T regulatory and [CD45.sup.+] [CD11b.sup.+] [CD103.sup.+] cells compared to control, two factors mediating OT. Mice given fg-Si[O.sub.2] exhibited intestinal Lcn-2 level and interferon gamma (IFN-[gamma]) secretion, showing inflammation and less production of IL-10 and TGF-[beta]. These effects were also observed in OVA-tolerized mice exposed to fg-Si[O.sub.2], in addition to a breakdown of OT and a lower intestinal frequency of T cells. In NOD/DQ8 mice immunized with gluten, the villus-to-crypt ratio was decreased while the [CD3.sup.+] intraepithelial lymphocyte counts and the Th1 inflammatory response were aggravated after fg-Si[O.sub.2] treatment. Discussion: Our results suggest that chronic oral exposure to fg-Si[O.sub.2] blocked oral tolerance induction to OVA, and worsened gluten-induced immunopathology in NOD/DQ8 mice. The results should prompt investigation on the link between Si[O.sub.2] exposure and food sensitivities in humans. https://doi.org/10.1289/EHP12758, Introduction Adverse food reactions include food sensitivities, which are immune mediated, and food intolerance, which is the result of failure to digest the triggering agent, as in lactose intolerance. (1,2) [...]

Acute myeloid leukemia (AML) presents a pressing medical need in that it is largely resistant to standard chemotherapy as well as modern therapeutics, such as targeted therapy and immunotherapy, including anti-programmed cell death protein (anti-PD) therapy. We demonstrate that programmed death-1 homolog (PD-1H), an immune coinhibitory molecule, is highly expressed in blasts from the bone marrow of AML patients, while normal myeloid cell subsets and T cells express PD-1H. In studies employing syngeneic and humanized AML mouse models, overexpression of PD-1H promoted the growth of AML cells, mainly by evading T cell-mediated immune responses. Importantly, ablation of AML cell-surface PD-1H by antibody blockade or genetic knockout significantly inhibited AML progression by promoting T cell activity. In addition, the genetic deletion of PD-1H from host normal myeloid cells inhibited AML progression, and the combination of PD- 1H blockade with anti-PD therapy conferred a synergistic antileukemia effect. Our findings provide the basis for PD- 1H as a potential therapeutic target for treating human AML., Introduction Acute myeloid leukemia (AML) is a heterogenous clonal disorder that is characterized by uncontrolled clonal expansion of myeloid progenitor cells (blasts) that leads to BM failure (1). AML is [...]

Foxp3-expressingTregs employ multiple suppressive mechanisms to curtail conventional T cell (Tconv) responses and establish tissue homeostasis. How Foxp3 coordinates Treg contact-dependent suppressive function is not fully resolved. In this issue of the JCI, Wang and colleagues revealed that Foxp3-mediated inhibition of ryanodine receptor 2 (RyR2) led to strong Treg-DC interactions and enhanced immunosuppression. RyR2 depletion in Tconvs phenocopied this effect and equipped Tconvs with Treg-like suppressive function in multiple inflammatory or autoimmune contexts. This study provides molecular and therapeutic insights underlying how cell-cell contact limits immune reactivity., Treg suppressive mechanisms limit immune reactivity Tregs are a specialized subset of [CD4.sup.+] T cells characterized by the expression of the master transcription factor, Foxp3, which orchestrates Treg development and [...]

A20 is a ubiquitin-modifying protein that negatively regulates NF-[kappa]B signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. [A20.sup.[DELTA]CNS-EC] mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS- ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of [A20.sup.[DELTA]CNS-EC] mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation., Introduction Multiple sclerosis (MS) is the most prevalent chronic inflammatory disease of the central nervous system (CNS) in young adults (1). To date, the causes of MS are not fully [...]

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine [CD8.sup.+] T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate ([Tex.sup.int]) and effector ([Tex.sup.eff]) exhausted T cell subsets derived from progenitor exhausted T cells ([Tex.sup.prog]) in tumors. A comprehensive CRISPR tiling screen of the 50C57-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies., Introduction Cell therapies using T lymphocytes possessing antitumor specificity have demonstrated robust clinical activity in hematological malignancies (1-4), with more limited success observed in solid tumors (5-7). The reduced effectiveness [...]

Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations in the autoimmune regulator (AIRE) gene. Most patients present with severe chronic mucocutaneous candidiasis and organ- specific autoimmunity from early childhood, but the clinical picture is highly variable. AIRE is crucial for negative selection of T cells, and scrutiny of different patient mutations has previously highlighted many of its molecular mechanisms. In patients with a milder adult-onset phenotype sharing a mutation in the canonical donor splice site of intron 7 (c.879+1G>A), both the predicted altered splicing pattern with loss of exon 7 ([Aire.sup.Ex7-/-]) and normal full-length AIRE mRNA were found, indicating leaky rather than abolished mRNA splicing. Analysis of a corresponding mouse model demonstrated that the [Aire.sup.Ex7-/-] mutant had dramatically impaired transcriptional capacity of tissue-specific antigens in medullary thymic epithelial cells but still retained some ability to induce gene expression compared with the complete loss-of-function [Aire.sup.C313X-/-] mutant. Our data illustrate an association between AIRE activity and the severity of autoimmune disease, with implications for more common autoimmune diseases associated with AIRE variants, such as primary adrenal insufficiency, pernicious anemia, type 1 diabetes, and rheumatoid arthritis., Introduction Autoimmune polyendocrine syndrome type 1 (APS-1) or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) (OMIM #240300) is a rare, childhood-onset disorder caused by mutations in the autoimmune regulator (AIRE) gene (1-4). It [...]

Regulatory T cells (Tregs) are instrumental in maintaining immune tolerance and preventing destructive autoimmunity, but how heterogeneous Treg populations are established remains largely unknown. Here, we show that Zfp335 deletion in Tregs failed to differentiate into effector Tregs (eTregs) and lose Treg-suppressive function and that KO mice exhibited early-onset lethal autoimmune inflammation with unrestricted activation of conventional T cells. Single-cell RNASeq analyses revealed that Zfp335-deficient Tregs lacked a eTreg population and showed dramatic accumulation of a dysfunctional Treg subset. Mechanistically, Zfp335-deficient Tregs displayed reduced oxidative phosphorylation and dysfunctional mitochondrial activity. Further studies revealed that Zfp335 controlled eTreg differentiation by regulating fatty acid oxidation (FAO) through direct targeting of the FAO enzyme Hadha. Importantly, we demonstrate a positive correlation between ZNF335 and HADHA expression in human eTregs. Our findings reveal that Zfp335 controls FAO-driven eTreg differentiation to establish immune tolerance., Introduction Regulatory T cells (Tregs) expressing forkhead box P3 (Foxp3) play a key role in the establishment and maintenance of peripheral immune tolerance through the prevention and suppression of autoimmunity [...]

Carcinogen exposure is strongly associated with enhanced cancer immunogenicity. Increased tumor mutational burden and resulting neoantigen generation have been proposed to link carcinogen exposure and cancer immunogenicity. However, the neoantigen-independent immunological impact of carcinogen exposure on cancer is unknown. Here, we demonstrate that chemical carcinogen-exposed cancer cells fail to establish an immunosuppressive tumor microenvironment (TME), resulting in their T cell-mediated rejection in vivo. A chemical carcinogen-treated breast cancer cell clone that lacked any additional coding region mutations (i.e., neoantigen) was rejected in mice in a T cell-dependent manner. Strikingly, the coinjection of carcinogen- and control-treated cancer cells prevented this rejection, suggesting that the loss of immunosuppressive TME was the dominant cause of rejection. Reduced M-CSF expression by carcinogen-treated cancer cells significantly suppressed tumor-associated macrophages (TAMs) and resulted in the loss of an immunosuppressive TME. Single-cell analysis of human lung cancers revealed a significant reduction in the immunosuppressive TAMs in former smokers compared with individuals who had never smoked. These findings demonstrate that carcinogen exposure impairs the development of an immunosuppressive TME and indicate a novel link between carcinogens and cancer immunogenicity., Introduction The advent of immune checkpoint blockade (ICB) therapy has greatly improved cancer treatment, especially for metastatic melanoma, non-small cell lung cancer and squamous cell carcinoma of the head and [...]

HIV-1 persists in a latent reservoir in resting [CD4.sup.+] T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART ([t.sub.1/2] = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent provi ruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with [t.sub.1/2] of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4* T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART., Introduction Antiretroviral therapy (ART) blocks HIV-1 replication, reducing plasma virus levels to below the detection limit of clinical assays (1-3). ART prevents disease progression but does not eliminate the latent [...]