T-cell Anergy Research Articles

Treatment Options for Patients with Non-Small Cell Lung Cancer and Liver Metastases

Lung cancer represents the most common cause of cancer-related death. Patients with non-small cell lung cancer (NSCLC) and liver metastases have worse prognosis, with an overall survival (OS) from three to six months. The majority of them have a poor response to chemotherapy, and the data are controversial regarding the response to immunotherapy. This could be because the liver is considered to be an immune-tolerant organ, which is characterized by T-cell anergy and immunosuppressive signals. This review evaluates current treatment options for patients with NSCLC and liver metastases. Combination therapies might be a better treatment option for this subgroup of patients. The addition of radiotherapy to immunotherapy could also be an option in selected patients. The resection of single liver metastasis should also be considered.

A B7-H3–Targeted CD28 Bispecific Antibody Enhances the Activity of Anti–PD-1 and CD3 T-cell Engager Immunotherapies

Abstract T-cell activation is a multistep process requiring T-cell receptor engagement by peptide–MHC complexes (Signal 1) coupled with CD28-mediated costimulation (Signal 2). Tumors typically lack expression of CD28 ligands, so tumor-specific Signal 1 (e.g., neoepitope presentation) without costimulation may be ineffective or even induce T-cell anergy. We designed the bispecific antibody XmAb808 to co-engage the tumor-associated antigen B7-H3 with CD28 to promote T-cell costimulation within the tumor microenvironment. XmAb808 costimulation was measured by its ability to activate and expand T cells and enhance T cell–mediated cancer cell killing in cocultures of human peripheral blood mononuclear cells and cancer cells and in mice engrafted with human peripheral blood mononuclear cells and tumor xenografts. XmAb808 avidly bound cancer cells and stimulated IL2 and IFNγ secretion from T cells cocultured with cancer cells engineered to deliver Signal 1 to T cells via a surface-expressed anti-CD3 antibody. XmAb808 enhanced expression of the antiapoptotic factor Bcl-xL and CD25, promoting survival and IL2-dependent expansion of T cells coupled with increased T cell–mediated cytotoxicity in vitro. XmAb808 combined with an EpCAM×CD3 bispecific antibody to enhance target cell killing through IL2-dependent expansion of CD25+ T cells. This combination also suppressed pancreatic tumor xenograft growth in mice. Furthermore, XmAb808 combined with an anti–programmed cell death protein 1 antibody to suppress breast tumor xenograft growth in mice. XmAb808 as monotherapy and in combination with an anti–programmed cell death protein 1 antibody is currently in clinical development in patients with advanced solid tumors. Our results suggest that XmAb808 may also combine with tumor antigen–targeted anti-CD3 (Signal 1) T-cell engagers.

Update on the Mechanism of Action of Intravesical BCG Therapy to Treat Non-Muscle-Invasive Bladder Cancer.

While more than four decades have elapsed since intravesical Bacillus Calmette-Guérin (BCG) was first used to manage non-muscle invasive bladder cancer (NMIBC), its precise mechanism of anti-tumor action remains incompletely understood. Besides the classic theory that BCG induces local (within the bladder) innate and adaptive immunity through interaction with multiple immune cells, three new concepts have emerged in the past few years that help explain the variable response to BCG therapy between patients. First, BCG has been found to directly interact and become internalized within cancer cells, inducing them to act as antigen-presenting cells (APCs) for T-cells while releasing multiple cytokines. Second, BCG has a direct cytotoxic effect on cancer cells by inducing apoptosis through caspase-dependent pathways, causing cell cycle arrest, releasing proteases from mitochondria, and inducing reactive oxygen species-mediated cell injury. Third, BCG can increase the expression of programmed death ligand 1 (PD-L1) on both cancer and infiltrating inflammatory cells to impair the cell-mediated immune response. Current data has shown that high-grade recurrence after BCG therapy is related to CD8+ T-cell anergy or 'exhaustion'. High-field cancerization and subsequently higher neoantigen presentation to T-cells are also associated with this anergy. This may explain why BCG therapy stops working after a certain time in many patients. This review summarizes the detailed immunologic reactions associated with BCG therapy and the role of immune cell subsets in this process. Moreover, this improved mechanistic understanding suggests new strategies for enhancing the anti-tumor efficacy of BCG for future clinical benefit.

Prognostic Effectiveness of PD-L1 Tumoral Expression in Oral Cavity Squamous Cell Carcinoma.

PD-L1 is a glycoprotein present on antigen-presenting cells and malignant cells and activates the PD-L1 receptor on cells surfaces, leading to T-cell anergy and death. The objective of this study was to assess PD-L1 tumoral expression in 50 patients with oral squamous cell carcinoma, define its prognostic value, and investigate its association with 2-year overall survival, locoregional recurrence, distant metastasis, and a number of other clinicopathological parameters. In a 24-month prospective observational study, involving 50 oral cavity squamous cell carcinoma patients, PD-L1 tumoral expression was evaluated using semiquantitative immunohistochemistry analysis by an experienced pathologist. PD-L1 expression of ≥ 6% of tumor cells was associated with poor survival outcome and determined to be a pertinent clinical cutoff value. PD-L1 expression of ≥ 6% was significantly associated with a number of clinicopathological parameters in our study of 50 patients with OSCC. Pathological nodal staging (P = 0.00), lympho-vascular invasion (P = 0.03), extra-nodal extension (P = 0.04), overall staging (P = 0.01), locoregional recurrence (P = 0.00), and distant metastasis (P = 0.00) all showed statistically significant associations. Our study concluded a significant correlation with decreased 2-year overall survival and 2-year disease free survival by using the Kaplan- Meier survival plot for overall survival (p = 0.02) and (p = 0.0002), respectively. The presence of ≥ 6% PD-L1 (CD274) tumoral expression was found to be significantly associated with 2-year overall survival (OS), locoregional recurrence (LRC), distant metastasis (DM), and various clinicopathological parameters. Tumoral PD-L1 was found as a discrete prognostic biomarker which showed significant association with tumor aggressiveness.

RNA-sequencing of Human Kidney Allografts and Delineation of T-Cell Genes, Gene Sets, and Pathways Associated With Acute T Cell-mediated Rejection.

Delineation of T-cell genes, gene sets, pathways, and T-cell subtypes associated with acute T cell-mediated rejection (TCMR) may improve its management. We performed bulk RNA-sequencing of 34 kidney allograft biopsies (16 Banff TCMR and 18 no rejection [NR] biopsies) from 34 adult recipients of human kidneys. Computational analysis was performed to determine the differential intragraft expression of T-cell genes at the level of single-gene, gene set, and pathways. T-cell signaling pathway gene sets for plenary T-cell activation were overrepresented in TCMR biopsies compared with NR biopsies. Heightened expression of T-cell signaling genes was validated using external TCMR biopsies. Pro- and anti-inflammatory immune gene sets were enriched, and metabolism gene sets were depleted in TCMR biopsies compared with NR biopsies. Gene signatures of regulatory T cells, Th1 cells, Th2 cells, Th17 cells, T follicular helper cells, CD4 tissue-resident memory T cells, and CD8 tissue-resident memory T cells were enriched in TCMR biopsies compared with NR biopsies. T-cell exhaustion and anergy were also molecular attributes of TCMR. Gene sets associated with antigen processing and presentation, and leukocyte transendothelial migration were overexpressed in TCMR biopsies compared with NR biopsies. Cellular deconvolution of graft infiltrating cells by gene expression patterns identified CD8 T cell to be the most abundant T-cell subtype infiltrating the allograft during TCMR. Our delineation of intragraft T-cell gene expression patterns, in addition to yielding new biological insights, may help prioritize T-cell genes and T-cell subtypes for therapeutic targeting.

Abstract 5558: Whole-slide multiplexed tissue imaging of prostate adenocarcinoma reveals distinct immune niches in high- versus low-grade tumors

Abstract Objectives Tumor-immune interactions are a critical determinant of tumor evolution and are increasingly targeted by novel therapeutics (e.g. immune checkpoint blockade). However, their significance in treatment-naive localized prostate cancer remains largely unknown. Here, we employ multiplexed tissue imaging on radical prostatectomy specimens across Gleason grades to create a single-cell spatial atlas of primary prostate tumors and characterize their innate and adaptive immune landscape. Methods Whole-slide radical prostatectomy (RP) specimens were obtained from 28 patients: 14 with Gleason Grade Group ≤ 3 (Low-grade; LG) and 14 with Gleason Grade Group ≥ 4 (High-grade; HG). Tumors were imaged with our cyclic immunofluorescence (CyCIF) platform using a 27-marker panel encompassing epithelial, T-cell, innate immune and stromal markers. Tumor and stromal compartments were manually annotated by a pathologist. Cell densities were compared between tumor and stromal compartments of LG and HG tumors. Results Across the 28 RP specimens, we identified and spatially resolved over 27 million individual cells. Overall, higher Gleason grade was associated with a significantly increased innate and adaptive immune reaction. CD8+ cytotoxic T-cell density was significantly higher in HG vs LG (p = 0.003) specimens, although this effect was largely due to enrichment in adjacent stroma rather than direct tumor infiltration. By contrast, FOXP3+ T-cells were significantly increased throughout the tumor and stromal compartments of HG compared with LG tumors (p = 0.001). Tertiary lymphoid structures (TLS) and CD163+ myeloid cells were also significantly enriched in HG tumors relative to LG tumors (p = 0.029 and 0.039, respectively). PD-1+ cells were specifically enriched in TLS structures in HG tumors (p < 0.001). There were no significant differences in CD11c+ and CD103+ cell densities between LG and HG tumors. Conclusions This spatially resolved single-cell atlas of localized prostate cancer reveals that high-grade tumors exhibit significant inflammation, particularly of TLSs and stromal CD8+, and are enriched in markers of T-cell anergy and myeloid-derived suppression. These findings raise the hypothesis that immunotherapy may have activity in treatment-naïve high-grade prostate cancer, despite the failure of immune checkpoint blockade in mCRPC. Citation Format: Jeremiah Wala, Jia-Ren Lin, Brian Labadie, Daniel Peiffer, Andreánne Gagne, Kiranj Chaudagar, Yu-An Chen Chen, Eliezer Van Allen, Peter Sorger, Akash Patnaik. Whole-slide multiplexed tissue imaging of prostate adenocarcinoma reveals distinct immune niches in high- versus low-grade tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5558.

E3 ubiquitin ligase casitas B-lineage lymphoma-b modulates T-cell anergic resistance via phosphoinositide 3-kinase signaling in patients with immune thrombocytopenia

BackgroundThe E3 ubiquitin ligase casitas B-lineage lymphoma-b (CBLB) is a newly identified component of the ubiquitin-dependent protein degradation system and is considered an important negative regulator of immune cells. CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms. However, the involvement of CBLB in the pathogenesis of immune thrombocytopenia (ITP) is unknown. ObjectivesWe aimed to investigate the expression and role of CBLB in CD4+ T cells obtained from patients with ITP through quantitative proteomics analyses. MethodsCD4+ T cells were transfected with adenoviral vectors overexpressing CBLB to clarify the effect of CBLB on anergic induction of T cells in patients with ITP. DNA methylation levels of the CBLB promoter and 5′ untranslated region (UTR) in patient-derived CD4+ T cells were detected via MassARRAY EpiTYPER assay (Agena Bioscience). ResultsCD4+ T cells from patients with ITP showed resistance to anergic induction, highly activated phosphoinositide 3-kinase–protein kinase B (AKT) signaling, decreased CBLB expression, and 5′ UTR hypermethylation of CBLB. CBLB overexpression in T cells effectively attenuated the elevated phosphorylated protein kinase B level and resistance to anergy. Low-dose decitabine treatment led to significantly elevated levels of CBLB expression in CD4+ T cells from 7 patients showing a partial or complete response. ConclusionThese results indicate that the 5′ UTR hypermethylation of CBLB in CD4+ T cells induces resistance to T-cell anergy in ITP. Thus, the upregulation of CBLB expression by low-dose decitabine treatment may represent a potential therapeutic approach to ITP.

Englumafusp Alfa (CD19-4-1BBL) and Glofitamab Combination in Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (R/R NHL): Biomarker Results from a Phase I Dose-Escalation Trial

Background: Englumafusp alfa is an antibody-like fusion protein that simultaneously targets CD19 on B cells and 4-1BB on T cells and other immune cells. In the presence of a T-cell receptor signal and strictly dependent on CD19 crosslinking, englumafusp alfa co-stimulates T cells via 4-1BB agonism that boosts T-cell effector functions and prevents T-cell anergy. An ongoing Phase I dose-escalation study (NCT04077723) is investigating the safety, efficacy, pharmacokinetics, and pharmacodynamics (PD) of intravenous administration of englumafusp alfa in combination with glofitamab in patients (pts) with R/R NHL. We recently presented preliminary clinical data demonstrating promising clinical activity and good tolerability in R/R NHL (Hutchings et al, ICML 2023). Here, we report preliminary peripheral blood (PB) and tissue biomarker analyses to demonstrate mechanisms of action (MoA), dose relationship, and baseline features associated with response. Methods: Exploratory biomarker analyses included data from 84 pts with indolent or aggressive NHL (aNHL) dosed with 0.36-75mg englumafusp alfa, starting on Cycle 2 Day 8 (C2D8) and followed by combination with the fixed target dose of glofitamab (30mg), once every 3 weeks, from C3D1 for up to 12 cycles. Immune profiling of PB by flow cytometry and plasma cytokines analysis by ELLA were performed. Circulating tumor DNA (ctDNA) dynamics were evaluated by the Avenio NHL CAPP-Seq assay (Stokowski et al, ASH 2022) at C3D1, C5D1, and the end of 12-cycle treatment (EoT) in patients with aNHL. Baseline tumor biopsies (n=65) were analyzed by immunohistochemistry/immunofluorescence assays. On-treatment (OT) PD changes during the first five cycles of treatment were evaluated and compared to historical glofitamab monotherapy PD data generated in a phase I/II study (NCT03075696). Results: Englumafusp alfa in combination with glofitamab induced PD changes in PB at all tested doses. Specifically, in a dose-dependent manner, englumafusp alfa in combination with glofitamab limited the PB expansion of fully differentiated PD1+ CD8+ effector memory T cells re-expressing CD45RA (Temra), previously shown to expand dose-dependently following glofitamab monotherapy (Broeske et al, Blood Advances 2022). Additionally, the combination also resulted in a significant PB expansion of the activated (HLA-DR+) and effector memory CD8+ T (T EM) cells, and the ratio of T EM to central memory and naïve CD8+ T cells compared to glofitamab monotherapy. Furthermore, we detected no significantly added cytokine release with the combination to that previously observed with glofitamab monotherapy, in line with the clinically observed safety profile. Preliminary analysis of ctDNA dynamics showed that at C3D1 33% (18/55), at C5D1 40% (15/38), and at EoT 53% (9/17) of the pts had no detectable ctDNA. Notably, 61% of pts with aNHL who achieved complete metabolic response (CMR) had no detectable ctDNA at any OT visit. Our preliminary analysis also indicated a significant (p=0.03) association between the observed OT expansion of CD8+ T EM cells and the extent of decrease in ctDNA levels (mean mutant molecules per milliliter) at EoT relative to baseline. Finally, we observed ~60% of pts with low tumor infiltrating CD8+ T cells (<500 cells/mm 2 CD8+) and a more aggressive disease (>5000 cells/mm 2 proliferative (Ki67+) tumor cells) at baseline, both poor outcome correlates of glofitamab monotherapy, achieved a CMR with higher doses of englumafusp alfa. Conclusions: In this study, we demonstrated the MoA of englumafusp alfa in R/R NHL and key PD effects in combination versus glofitamab monotherapy that will support optimal biological dose finding. Furthermore, the observed association between expansion of T EM cells in PB and changes in ctDNA dynamics at EoT, albeit preliminary, supports ctDNA as a marker for depth and durability of response. Overall, our PD and biomarker observations so far strengthen the rationale of combining glofitamab with englumafusp alfa to drive long-term responses in this heavily pre-treated NHL patient population. Acknowledgments: This study was sponsored by F. Hoffmann-La Roche Ltd. The authors would like to thank the patients who participated in this trial, their families, and their caregivers. Special thanks also to Daria Rukina, Iva Lelios, Philip Knuckles, Kat Reyskens, Grigori Singovski, and Joanne Hayward for their contributions to this work.

APLs and Oranges: Induction of T Cell Anergy by Altered Peptide Ligands.

This Pillars of Immunology article is a commentary on “Induction of T-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cells,” a pivotal article written by J. Sloan-Lancaster, B. D. Evavold, and P. M. Allen, and published in Nature, in 1993. https://www.nature.com/articles/363156a0

Differential Effects of Anti-PD-1/PD-L1 Checkpoint Inhibitors on Adhesion Molecules and Cytokine Secretion by THP-1 Monocytes.

Immune checkpoint inhibitors have improved the treatment regimen for human cancers in recent years. Particularly, inhibitors of the checkpoint molecules PD-1/PD-L1 have emerged as promising therapeutic treatments by preventing T-cell anergy and exhaustion. However, the impact of different anti-PD-1/PD-L1 checkpoint inhibitors on human monocytes remains elusive. In this study, using the human monocyte leukemia cell line THP-1 as a model, we investigated the influence of different therapeutic anti-PD-1/PD-L1 checkpoint inhibitors on monocytic adhesion molecule expression and cytokine secretion. THP-1 monocytes were treated with the anti-PD-1 checkpoint inhibitors Nivolumab and Pembrolizumab and anti-PD-L1 checkpoint inhibitors Atezolizumab and Durvalumab. Cytokine expression patterns were evaluated using cytokine arrays and enzyme-linked immunosorbent assays (ELISA) and analysis of adhesion molecules was addressed using flow cytometry. Our data show an overall moderate apoptosis induction upon checkpoint inhibitor treatment and significantly reduced expression levels of adhesion molecules CD29, CD49d, and CX3CR1 in response to anti-PD-1 treatment. Cytokine screening revealed overall decreased secretion levels of insulin-like growth factor binding protein 2 (IGFBP2), CD147 (basigin) and CD31 (PECAM-1) as well as elevated levels of interleukin 5 (IL-5) and interferon gamma (IFNγ) in response to checkpoint inhibitor treatment. Our data indicate differential effects of anti-PD-1/PD-L1 checkpoint inhibitors on THP-1 monocytes, both by specific anti-PD-1/PD-L1 binding and unspecific antibody IgG isotype recognition. Further investigations on peripheral blood monocyte subsets in terms of their expansion and function upon checkpoint inhibitor therapy are required to better understand the individual immunological balances in cancer patients in long-term observational studies.

A single-amino acid substitution in the adaptor LAT accelerates TCR proofreading kinetics and alters T-cell selection, maintenance and function

Mature T cells must discriminate between brief interactions with self-peptides and prolonged binding to agonists. The kinetic proofreading model posits that certain T-cell antigen receptor signaling nodes serve as molecular timers to facilitate such discrimination. However, the physiological significance of this regulatory mechanism and the pathological consequences of disrupting it are unknown. Here we report that accelerating the normally slow phosphorylation of the linker for activation of T cells (LAT) residue Y136 by introducing an adjacent Gly135Asp alteration (LATG135D) disrupts ligand discrimination in vivo. The enhanced self-reactivity of LATG135D T cells triggers excessive thymic negative selection and promotes T-cell anergy. During Listeria infection, LATG135D T cells expand more than wild-type counterparts in response to very weak stimuli but display an imbalance between effector and memory responses. Moreover, despite their enhanced engagement of central and peripheral tolerance mechanisms, mice bearing LATG135D show features associated with autoimmunity and immunopathology. Our data reveal the importance of kinetic proofreading in balancing tolerance and immunity.

Manifestations of B-cell expansion with NF-κB and T-cell anergy disease overlapping with hemophagocytic lymphohistiocytosis.

B cell expansion with NF-κB and T cell anergy (BENTA) is a disease genetically linked with heterozygous gain-of-function (GOF) mutations in the CARD11 gene with an autosomal dominant expression. Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous group of disorders characterized by systemic inflammation and hypercytokinaemia. Some BENTA patients share similar clinical manifestations as HLH in various aspects, including feverand splenomegaly. In this study, we reported a 15-month-old boy diagnosed as BENTA meeting with diagnostic criteria of HLH. The complications were resolved by antibiotics for controlling severe infection, together with the reduced format of dexamethasone and etoposide for subsiding HLH activities. While the patient was not subjected to disease recurrence and maintained free of infection, a persistent lymphocytosis derived mainly from the expansion of polyclonal B cells was ascertained. Flow cytometry analysis indicated that the subdued degranulation of NK cells prior to treatment had been restored as the HLH-related complications waned. With largely reduced number and ratios in CD4 and CD8 T cells, their proliferation and Vβ diversity remained in normal ranges. In vitro stimulation experiment revealed a functional abbreviation of T cells as the percentage of IFNγ-releasing CD3+CD4+ T cells augmented while the percentage reduced in CD3+CD4- T cells. Whole exome sequencing revealed a de novo G123D missense mutation in the CARD11 gene. This new case of BENTA showcased a scenario of predominant HLH activities accompanied by a severe infection normally associated with BENTA. In addition, a brief treatment quenching HLH complication in cooperation with antibiotics for infection control was not able to solve the underlined T cell abnormality as well as B cell expansion caused by CARD11 mutation. A haematopoietic stem cell transplantation or gene therapy is still a pursuit to remedy this inborn error of immunity.

Abstract P052: Carbidopa, an activator of aryl hydrocarbon receptor, suppresses IDO1 expression in pancreatic cancer and decreases tumor growth

Abstract IDO1 is an immunomodulatory enzyme responsible for tryptophan catabolism. Its expression in immune cells, especially the DCs, has attracted attention because it leads to tryptophan depletion at the immunological synapse, thereby causing T-cell anergy and immune evasion by the tumor cells. Cancer cells also overexpress IDO1. Immunotherapy targeting IDO1 has been one of the focus areas in cancer biology, but lately studies have identified non-immune related functions of IDO1 leading to a paradigm shift about IDO1 function in the context of tumor cells. In this study, we show that PDAC tissues and PDAC cells overexpress IDO1. The expression level is reciprocally related to overall patient survival. We further show that carbidopa, an FDA-approved drug for Parkinson’s disease as well as an AhR agonist, inhibits IDO1 expression in PDAC cells. Using athymic nude mice, we demonstrate that carbidopa-mediated suppression of IDO1 expression attenuates tumor growth. Mechanistically, we show that AhR is responsible for carbidopa-mediated suppression of IDO1, directly as a transcription factor and indirectly by interfering with the JAK/STAT pathway. Overall, targeting IDO1 not only in immune cells but also in cancer cells could be a beneficial therapeutic strategy for PDAC and potentially for other cancers as well and that carbidopa could be repurposed to treat cancers that overexpress IDO1. Citation Format: Ksenija Korac, Yangzom D Bhutia, Devaraja Rajasekaran. Carbidopa, an activator of aryl hydrocarbon receptor, suppresses IDO1 expression in pancreatic cancer and decreases tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P052.

A case report involving suppressed nuclear receptor transcription factors 4a1 and Stevens-Johnson syndrome induced by a single dose of pembrolizumab and successfully treated with early steroid administration, resulting in complete remission of stage III lung cancer

BackgroundImmunotherapy with immune checkpoint inhibitors is associated with immune-related adverse events (irAEs). A positive correlation between treatment efficacy and irAEs has been reported. Clinical indicators are required for appropriate interventions, such as steroid administration, to prevent fatal outcomes. Nuclear receptor transcription factor 4a (Nr4a), which is involved in T-cell anergy, exhaustion, and regulatory T cells, were observed not only in thymocytes but in peripheral blood mononuclear cells. We describe a case of Stevens-Johnson syndrome (SJS) that was induced by a single dose of pembrolizumab and successfully treated with steroids, leading to complete remission of lung cancer during the monitoring of immune response indices, including Nr4a1 mRNA.Case presentationA 68-year-old male with squamous cell lung cancer (cT2aN3M0, stage IIIb) received a single dose of pembrolizumab (200 mg). On Day 21 of treatment, SJS appeared, and the patient was treated with prednisolone 60 mg/day, which was gradually tapered off. After the disappearance of the SJS symptoms, complete remission of cancer was achieved and was maintained for more than 1 year. Acute increases in the plasma IFN-γ and IL-17 concentrations and a decrease in IL-10 concentrations were observed at the onset of SJS. Simple regression analysis showed that these changes in IL-17, IFN-γ and IL-10 were significantly influenced by the decreased expression of Nr4a1 mRNA. The pembrolizumab levels and prednisolone doses significantly influenced the suppression of Nr4a1 mRNA levels. Although Nr4a1 mRNA levels in the current case fluctuated during the observation period, they were significantly lower than those in a nonresponding progressive-disease case, as well as a pembrolizumab-responding case with non-SJS but similar background. The suppression of Nr4a1 in current case, might result in upregulation of cytotoxic T cells and a reduction in functional regulatory T cells, promoting favorable antitumor immunity.ConclusionThe immune responses involving Nr4a1 suppression might relate to complete remission of lung cancer in this case, despite causing SJS, which may be attributed to synergistic effects from pembrolizumab treatment and intervention with steroids. The current case indicates the preliminarily clinical benefit of evaluating Nr4a expression-related indices as the possible clinical covariates and may serve as a milestone for appropriate future chemotherapy interventions.

Progressive Multifocal Leukoencephalopathy Associated With Sarcoidosis: A Multi-Center Case Series

Objective We aim to describe the clinical, laboratory, and radiographic features that characterize patients with progressive multifocal leukoencephalopathy (PML) in the context of sarcoidosis (S-PML). Background Sarcoidosis has been associated with CD4+, CD8+, and CD19+ lymphopenia, T-cell anergy, and increased infection risk. S-PML has been reported in approximately 60 cases. PML is often mistaken for neurosarcoidosis, leading to harmful administration of high-dose steroids. Preliminary evidence suggests that experimental therapies such as interleukins 2 and 7, checkpoint inhibitors, polyomavirus-specific T-cell therapy, and infliximab may offer promise for treatment. To ensure optimal outcome, it is crucial to identify S-PML accurately and with minimal delay. Design/Methods Data and imaging for patients were collected retrospectively from the electronic medical record from Mass General-Brigham network hospitals, National Institutes of Health, Mayo Clinic, Columbia University Irving Medical Center, University of California San Francisco, University of Michigan, and Beth Israel Deaconess Medical Center. Results Twenty-five patients with definite S-PML were identified. Median age at diagnosis of sarcoidosis was 54 years, and median time between sarcoidosis and PML diagnosis was 12 months. Sarcoidosis was isolated to lung in 14/25 patients; 10/25 had multisystem involvement; one patient had isolated dermatologic sarcoidosis. Of all patients, 16/25 patients had never received immunosuppressive medications prior to neurological symptom onset. Median serum lymphocyte count at time of PML diagnosis was 430 cells/uL (range: 50-1490). On MRI, 8/25 patients had infratentorial lesions only, while 8/25 had both infratentorial and supratentorial lesions, and 6/25 showed contrast enhancement. Seven/twenty-five patients progressed to death. Conclusions This study characterizes the clinical, laboratory, and imaging features of S-PML patients from seven major US medical centers. These data will be used to identify risk factors for development of PML in the context of sarcoidosis and to investigate any biomarkers that might aid in accurate and timely diagnosis.

B-cell expansion with nuclear factor-κB and T-cell anergy disease treated with rituximab in a child

患儿8月龄起病以脾脏、淋巴结和腮腺肿大为突出表现，伴反复发热、窦肺感染、贫血和血小板减少，外周血多克隆性B细胞增殖。基因检出半胱天冬酶募集结构域11基因第5外显子新发杂合变异（c.368C>T，p.G123D），父母为野生型，诊断NF-κB相关B细胞增殖和T细胞失能性疾病。经10年利妥昔单抗治疗，淋巴增殖控制良好。.

CARD11 MUTATION IN A PATIENT WITH VERY EARLY ONSET IBD AND IMMUNODEFICIENCY

<h3>Background</h3> Caspase activation and recruitment domain 11 (CARD11) is a lymphocyte-specific scaffolding protein which plays are role in signaling through both the NF-κB and mTOR signaling cascades important in B- and T-cell function. Various mutations in the <i>CARD11</i> gene cause several different phenotypes. Heterozygous dominant negative (DN) mutations can result in variable immune deficiency, with associated predisposition for atopy. Gain-of-function mutations causes BENTA (B-cell Expansion with NF-κB and T-cell Anergy) disease. While most patients with <i>CARD11</i> mutations are diagnosed in childhood, some milder phenotypes can result delayed diagnosis. <h3>Case Description</h3> A 4-year-old female was diagnosed with VEO-IBD (ulcerative colitis). She had a history of recurrent infections since 8-months-old including infantile pneumonia, otitis media, MRSA skin abscesses, molluscum contagiosum and cutaneous warts, as well as eczema, allergic rhinitis and asthma. Immunologic workup revealed inadequate response to polysaccharide vaccine . Lymphocyte proliferation was decreased to <i>Candida</i> and Tetanus antigens. Despite IVIG infusions, with control of sinopulmonary infections but her viral cutaneous infections worsened. Her IBD was unresponsive to anti-TNF therapy, but improved with vedolizumab. Primary immunodeficiency diseases panel (426 gene) revealed a heterozygous <i>CARD11</i> VUS (c.223C>T, p.Arg75Trp). The same variant was present in her father, who was symptomatic with molluscum contagiousum and aphthous ulcers. <h3>Discussion</h3> We present a unique CARD11 mutation in the coiled-coil domain region of the CARD11 gene is suspected of causing a DN phenotype, including recurrent bacterial sinopulmonary and viral infections, atopic features, and VEO-IBD. In vitro functional studies of this mutation are in process to confirm pathogenicity of this variant.

VSTM5 is a novel immune checkpoint that promotes oral tolerance of cell-mediated and antibody responses

The V-set and transmembrane domain-containing protein (VSTM) family is a newly discovered immunoglobulin (Ig) superfamily that shares structural similarities with the B7-like transmembrane proteins. Although most VSTM5 members have been reported to exert immune-related functions, VSTM5 has been described as a regulator of neuronal morphogenesis and migration in the brain. Based on its close phylogenetic relationship with two immune checkpoints, VISTA and TIGIT, we investigated the potential role of VSTM5 in T-cell immune responses. VSTM5.Ig inhibits T-cell proliferation and cytokine production, induces T-cell apoptosis, and promotes the generation of regulatory T cells (Tregs) in in vitro T-cell assays. VSTM5 also contributes to the maintenance of T-cell anergy in vitro. Similarly, serum VSTM5.Ig produced using a recombinant plasmid in ovalbumin (OVA)-immunized mice inhibits both naive and effector T-cell immune responses. In addition, VSTM5.Ig enhances oral tolerance of cell-mediated and antibody responses in OVA-fed mice by inducing Tregs and T-cell clonal deletion. Consequently, our findings suggest that VSTM5 is a novel immune checkpoint that could be used to improve the therapeutic efficacy of tolerance-based therapies for autoimmune diseases.

A single administration of hIL-7-hyFc induces long-lasting T-cell expansion with maintained effector functions

AbstractInterleukin-7 (IL-7) is an essential cytokine for T-cell homeostatic proliferation and maintenance. Clinical studies have shown the potential benefits of IL-7 therapy in various diseases associated with lymphopenia. However, the kinetics of the T-cell response to a single administration of IL-7 in humans have not been fully elucidated. Here, we investigated the effects of Fc-fused long-acting recombinant human IL-7 (hIL-7-hyFc, efineptakin alfa) on lymphocytes in healthy adults after a single subcutaneous or intramuscular administration. Administration of hIL-7-hyFc increased the CD8+ and CD4+ T-cell numbers up to 2.5-fold, with corresponding upregulation of Ki-67 and Bcl-2 expression, peaking at day 3 or 7. Regulatory T cells (Tregs) did not expand. Among CD8+ and CD4+ T cells, all T-cell subsets (TN, TEM, TCM, TEMRA, and TSCM) increased for 56 days. The T-cell receptor repertoire diversity of naive CD8+ and CD4+ T cells was increased by hIL-7-hyFc, whereas the memory T-cell subsets did not differ between day 56 and day 0. Transcriptomic analysis revealed that hIL-7-hyFc induced robust T-cell expansion without changes in gene expression profiles associated with T-cell functions or genes related to T-cell exhaustion, senescence, and anergy. The effector functions of antigen-specific CD8+ T cells were preserved after hIL-7-hyFc administration. Our results suggest that hIL-7-hyFc administration induced a sustained increase in the numbers of CD8+ and CD4+ T cells, but not Tregs, without qualitative changes. These results support the potential of hIL-7-hyFc as a treatment for patients with compromised T-cell immunity or as a vaccine adjuvant.

P042: Metabolic regulation of adaptive response to arginine deprivation in Hodgkin Lymphoma

Our group has previously shown an altered aminoacidic homeostasis in the microenvironment of classic Hodgkin`s Lymphoma (HL), due to arginine deprivation, which plays a major role in the immune-escape mechanisms and T-cell anergy induction. To study the metabolic adaptive response to arginine deprivation in vitro, we cultured three HL human cell lines (L428, L540, KMH2) with customized complete media or lacking Arg (R0), supplemented with 10% dialyzed fetal bovine serum, in six independent experiments to collect their global metabolomic analysis by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc and transcriptome profiling by RNA-seq. Apotpostosis and mitochondrial depolarization were measured by FACS. Findings were validated by qRT-PCR and Western Blot analysis. Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements in KMH2 and HDMYZ cell lines, involving upregulation of genes required for the unfolded protein response, p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except for the genes involved in lactate metabolism. Arg deprivation caused mitochondrial distress and transcriptional reprogramming, via induction of oxidative stress, affecting the mitochondrial activity, switching from glucose-based metabolism to mitochondrial oxidation of fatty acids, requiring the transfer of fatty acids from lipid droplets to mitochondria. RNA-seq analysis showed that induction of ferroptosis key genes GPX4, TXNRD1 and ACSL4 together with reduced intracellular GSH levels and increased amount of cystine and methionine sulfoxide occurred upon arginine deprivation. The low-energy metabolic state induced by the adaptive response to arg-deprivation posed KMH2-HL cells into a quiescence state, with elevated HMOX1 and HMOX-2 to scavenge the excess ROS with subsequent genome instability as shown by increased γHA2X+/ATM+ cells. In HDMYZ, the increased oxidative stress due to arginine deprivation induced the engagement of the UFMylation pathway. These findings were confirmed in vivo, since in peripheral blood of HL patients we found reduced amount of GSH, glutamine and S-adenosylhomocysteine, citrate, methionine (p<0.001), as detected by HPLC. Taken together, our data suggest how arginine deprivation can regulate lipid trafficking, ferroptosis and UFMylation, novel potential targets to overcome drug resistance in cHL.