Immune checkpoint expression and relationships to anti-PD-L1 immune checkpoint blockade cancer immunotherapy efficacy in aged versus young mice

Metachronous malignancies after response to checkpoint inhibition

Immunotherapy in cancer is facing a revolution with the advent of immune-checkpoint inhibitors (ICI).

Simple SummaryTumor immune evasion is mediated in large part by the inhibition of anti-tumor T cell responses. Both the induction of tumor antigen-specific T cells and the activation state of T effector cells may be attenuated by surface receptors, which upon binding to counter receptors on immunoregulatory cell types and tumor cells, induce inhibitory T cell signaling. Immune checkpoint inhibitors (ICI) are antibodies that block interaction of these receptor pairs and thereby prevent T cell inhibition. Only a small percentage of tumor patients are responsive to treatment with ICI, which on the one hand raises the issue of possible reasons for failure, and on the other hand has spurred the development of additional ICI targeting other T cell inhibitory receptors. Our review aims to summarize knowledge on the functional role of these (inhibitory) receptors by additional types of leukocytes, and consequences of receptor blockade by ICI as a potential cause for unwanted side effects limiting the success of therapy. Deeper knowledge in this regard is a prerequisite for the development of more refined combination therapies.The approval of immune checkpoint inhibitors (ICI) that serve to enhance effector T-cell anti-tumor responses has strongly improved success rates in the treatment of metastatic melanoma and other tumor types. The currently approved ICI constitute monoclonal antibodies blocking cytotoxic T-lymphocyte-associated protein (CTLA)-4 and anti-programmed cell death (PD)-1. By this, the T-cell-inhibitory CTLA-4/CD80/86 and PD-1/PD-1L/2L signaling axes are inhibited. This leads to sustained effector T-cell activity and circumvents the immune evasion of tumor cells, which frequently upregulate PD-L1 expression and modulate immune checkpoint molecule expression on leukocytes. As a result, profound clinical responses are observed in 40–60% of metastatic melanoma patients. Despite the pivotal role of T effector cells for triggering anti-tumor immunity, mounting evidence indicates that ICI efficacy may also be attributable to other cell types than T effector cells. In particular, emerging research has shown that ICI also impacts innate immune cells, such as myeloid cells, natural killer cells and innate lymphoid cells, which may amplify tumoricidal functions beyond triggering T effector cells, and thus improves clinical efficacy. Effects of ICI on non-T cells may additionally explain, in part, the character and extent of adverse effects associated with treatment. Deeper knowledge of these effects is required to further develop ICI treatment in terms of responsiveness of patients to treatment, to overcome resistance to ICI and to alleviate adverse effects. In this review we give an overview into the currently known immunomodulatory effects of ICI treatment in immune cell types other than the T cell compartment.

Despite the considerable success of immune checkpoint therapies targeting T cells, a sizable proportion of patients experience resistance or relapse due to the immunosuppressive nature of the tumor microenvironment. Myeloid cells, a major component that suppresses effector lymphocytes, have emerged as an alternative and promising therapeutic target. However, there is a deep lack of widely accessible immunological models capable of representing the intricate three-way interaction between tumor cells, T cells, and myeloid cells. To address this need, we conducted a comprehensive protein profiling of human tumor and immune cell lines available at ATCC for various established and novel immune checkpoint molecules. Cell lines with high endogenous expression of the immune checkpoint proteins, such as programmed death-ligand 1 and 2 (PD-L1 and PD-L2), cluster of differentiation 155 (CD155), B7 homolog 3 (B7-H3), sialic acid-binding Ig-like lectin 10 (Siglec-10), or signal-regulatory protein alpha (SIRPα), were selected and constructed into luciferase reporter cell lines. For tumor reporter cell lines, a gamma interferon activation site (GAS) response element was placed upstream of the luciferase gene in the lentiviral vector, enabling the activation of the JAK-STAT signaling pathway within tumor cells to induce luciferase expression. In myeloid reporter cell lines, a nuclear factor kappa B (NF-κB) response element replaced GAS to monitor the activation of the NF-κB signaling pathway. In the presence of corresponding immune checkpoint inhibitors that enhance T cell-mediated anti-tumor activity, these reporter cell lines produce a bioluminescent signal based on luciferase expression. This signal can be easily detected and quantified to assess the efficacy of the inhibitor. Our data revealed that bioluminescence intensity in the tumor and myeloid reporter cell lines increased by >100-fold in a dose-dependent manner in response to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) stimulation, respectively, and by >50-fold in response to the conditioned media collected from activated primary T cells. Furthermore, in co-culture assays involving various combinations of immune cell and tumor cell types with corresponding immune checkpoint inhibitors, these reporter cell lines demonstrated a significant increase in bioluminescence intensity. In conclusion, these newly established luciferase reporter cell lines offer an excellent ex vivo model for cancer immunotherapy. These cell lines naturally express immune checkpoint proteins, enabling the sensitive and reproducible monitoring of combinatorial responses from various immune cell types. Citation Format: Hyeyoun Chang, Alicia C. Walker, John G. Foulke, Luping Chen, Fang Tian, Zhizhan Gu. Luciferase reporter cell lines allow simultaneous incorporation of tumor cells, innate immune cells, and adaptive immune cells for in-depth immune checkpoint studies [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 75.

8633 Background: Immune checkpoint inhibition (ICI) has significantly improved survival in patients (pts) with metastatic non-small cell lung cancer (NSCLC). While ICI duration is commonly limited to two years, the optimal duration of ICI and post-progression treatment outcomes in pts who received at least two years of ICI are unknown. Methods: We conducted an international, multi-center study of pts with metastatic NSCLC treated with ICI for at least two years to characterize clinical outcomes and post-progression treatment patterns. Log-rank tests were used to test for differences in event-time distributions, and Cox proportional hazards models were used to estimate HRs. Results: We identified 255 pts with metastatic NSCLC treated with ICI alone (N=210, 82.3%) or ICI + chemotherapy (N=45, 17.7%) for least two years. Median age was 63.0, 42.4% were women, 93.0% had history of tobacco use, 79.2% had adenocarcinoma histology, 61.4% had a PD-L1 TPS ≥50%. From the start of ICI, the median time to progression (mTTP) was 6.9 years (95%CI 4.9-NR) and the median overall survival (mOS) was 9.5 years (95%CI 8.0-NR). Pts wo achieved a complete/partial response to ICI had a significantly longer mTTP (HR 0.41, P<0.01) and mOS (HR 0.30, P<0.01) compared to those with a best objective response of stable disease. Patients with high PD-L1 ≥50% (HR 0.6, P=0.02 vs PD-L1 <50%), very high tumor mutational burden (TMB) ≥20 mut/Mb (HR 0.7, P=0.03, vs <20 mut/Mb) and ever smoking status (HR 0.52, P=0.04, vs never smokers) had also significantly longer mTTP. There was no difference in mTTP (HR: 0.98, P=0.94) and mOS (HR: 0.61, P=0.06) between pts who stopped ICI at two years of treatment (N=112) and those who continued ICI beyond two years (N=143). Among 96 pts who progressed after ICI, 33.3% (N=32) received local ablative therapies (surgery or radiation), 9.3% (N=9) pts received systemic chemotherapy and 31.2% (N=30) pts received ICI retreatment. Among those retreated with ICI, the response rate was 63%, mTTP and mOS from the date of start of ICI retreatment were 22.7 (95%CI 11.2-NR), and 41.7 months (19.3-NR), respectively. Clinico-genomic predictors of response to ICI retreatment included higher TMB at baseline (15 vs 7.6 mut/Mb, P<0.001), adenocarcinoma histology (94.4% vs 50.0%, P=0.01), and longer ICI treatment duration prior to retreatment (31.2 vs 24.5 months, P=0.02). Conclusions: Pts who received a minimum of two years of treatment with ICI experienced unprecedented long-term survival. There was no difference in outcomes between pts who stopped at two years compared to those who continue therapy. After completing two years of initial ICI therapy, a large fraction of pts who subsequently experience disease progression and are retreated with ICI experience an objective response, particularly those with longer duration of prior ICI treatment, higher TMB, and adenocarcinoma histology.

Cancer cell intrinsic TIM-3 induces glioblastoma progression

Drug-induced hypersensitivity syndrome like reaction with angioedema and hypotension associated with BRAF inhibitor use and antecedent immune checkpoint therapy

Simple SummaryPatients with high expression of CXCL8 are not sensitive to immune checkpoint inhibitors (ICIs) treatment, but the mechanism is unclear. LSECtin is the immune checkpoint ligand of LAG3, and is considered as an important factor of ICIs resistance. This study confirmed the role of CXCL8 and LSECtin in immune microenvironment modulation of colon cancer. The expression of CXCL8 is positively correlated with more than 40 immune checkpoints. CXCL8 could up-regulate LSECtin through AKT signal and promoted the proliferation and invasion ability of colon cancer. These results may be important reasons for the primary drug resistance of ICIs in colon cancer.Background: The role of CXCL8 and LSECtin in colon cancer liver metastasis and immune checkpoint inhibitors (ICIs) treatment effect were widely recognized. However, the regulatory role of CXCL8 on LSECtin is still unclear. Methods: The expression of CXCL8 or LSECtin was analyzed by TCGA database, and verified by GES110225 and clinical samples. The relationship between the expression of CXCL8 or LSECtin and immune cells infiltration, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, Gene Ontology (GO) items, stromal score, Estimation of STromal and Immune cells in MAlignant Tumours (ESTIMAT) immune score, tumor mutation burden (TMB), mismatch repair gene and immune checkpoints expression were analyzed by Spearman. The effects of CXCL8 on LSECtin expression, proliferation, and invasion ability were clarified by recombinant CXCL8 or CXCL8 interfering RNA. Results: In colon cancer, the expression of CXCL8 was higher, but LSECtin was lower than that in normal mucosa. The expression of CXCL8 or LSECtin was significantly positively correlated with immune cells infiltration, stromal score, ESTIMATE immune score, TMB, and immune checkpoints expression. The expression of LSECtin was closely related to the cytokine-cytokine receptor interaction pathway and response of chemokine function, such as CXCL8/CXCR1/2 pathway. There was a significant positive correlation between the expression of CXCL8 and LSECtin in colon cancer. CXCL8 up-regulated LSECtin through AKT signal and promoted the proliferation and invasion ability of colon cancer. Conclusions: CXCL8 up-regulated LSECtin by activating AKT signal and correlated with the immune microenvironment modulation in colon cancer.

Immunotherapy by immune checkpoint inhibitors (ICIs) revolutionized the treatment of melanoma patients. Tumor-infiltrating immune cells (TIICs) play a crucial role in antitumor immunity activated by ICIs. However, ICIs treatment may be associated with serious immune-related adverse events (irAEs). The aim of the study was to identify the key immune cells and molecules of the tumor microenvironment responsible for the treatment effects and risk of irAEs through immunohistochemical quantification of TIICs. We analyzed metastases (FFPE) of 28 melanoma patients treated with ICIs. Multilevel sampling and stereological quantification were used to assess TIICs identified immunohistochemically by the markers CD1a, CD1d, CD3, CD4, CD8, CD20, CD56, CD68, FOXP3, including immune checkpoint molecules LAG3, PD1, PD-L1. In lymph node metastases, higher infiltration of PD-L1, CD8-positive cells and lower infiltration of CD1a-positive cells predicted response to ICIs (P≤0.05, P≤0.05, P≤0.05, resp.). In all metastasis's subtypes, higher expression of PD-L1 was predictor of response to immunotherapy (P≤0.05). Lower PD-L1 expression (P≤0.05) and lower CD3 expression (P≤0.001) were associated with irAEs. Higher infiltration of CD8-positive T lymphocytes was associated with longer progression-free survival (P=0.0166) as well as overall survival (P=0.0454). Stereological quantification of specific immune cells in melanoma metastases, such as T-lymphocytes (CD3), cytotoxic T-lymphocytes (CD8), dendritic cells (CD1a) and PD-L1-positive cells, may predict ICIs treatment efficacy or the risk of irAEs. High infiltration of metastatic tissue by CD8-positive T cells is important for long-term favorable therapeutic response to ICIs.

An increasing number of therapy regimens using a combination of different immune checkpoint inhibitors (ICIs) have shown remarkable results in several different tumor entities. However, the likelihood of a positive response rate to combined ICIs is poor in most tumor entities and depends on several parameters including the tumor microenvironment. Particularly little is known about the spatial orchestration and spatial interplay between different immune checkpoint expressing cells. Given that the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is expressed on both immune cells as well as tumor cells and that several phase I/II studies are currently evaluating anti-TIM3 drugs, the interplay between these immune checkpoints in human cancers is of topical interest. To study the spatial orchestration and interplay between TIM3, CTLA-4, PD-1, and PD-L1 expression on T-cell subsets, macrophage subsets, CD11c+ dendritic cells, CD20+B-cells in relation to panCK+ malignant cells, CD31+ vessels and other structural tumor compartments, a multiplex fluorescence immunohistochemistry approach was used to stain 18 different antibodies on a set of tissue microarrays containing samples from more than 3000 carcinoma samples. In addition, a deep learning-based framework for cell type identification was developed and validated in this study. TIM3, PD-1, PD-L1, and CTLA-4 expression was measured on tumor cells (panCK+), cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+), subsets of macrophages (CD68+CD163+/CD68+iNOS+) and dendritic cells (CD11c+). Interestingly, TIM3 as well as CTLA-4 expression on CD3+CD8+ cytotoxic T-cells and CD3+CD4+FOXP3+ regulatory T-cells showed a spatially more diverse expression pattern - particularly inverse expression profile - compared to PD-1 expression on all analyzed T-cells subsets that was consistently accompanied by PD-L1 expression on immune and tumor cells (p<0.001). Combined analysis of cell densities, expression patterns, intensity measurements, interaction and distance analysis between immune cells and tumor cells revealed distinct changes in the immune cell infiltration pattern that was linked to several major immune checkpoint receptor expression profiles. Previously uncharacterized immune cell-composition dynamics in clustered tumor phenotypes, according to the immune checkpoint expression, were detected. This included for instance, a significant inverse association between CTLA-4 expression on T-cells and high expression levels of the PD-1/PD-L1 axis. In conclusion, deep profiling of 18 biomarkers in more than 40 different carcinoma entities revealed complex changes in the spatial orchestration of a wide range of immune cell subsets that was driven by the expression profile and composition of TIM3, PD-1, PD-L1, and CTLA-4. Citation Format: Nicolaus F. Debatin, Elena Bady, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Niclas C. Blessin. Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&STAIN mfIHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1935.

Objectives Patients with head and neck squamous cell carcinoma (HNSCC) have poor prognosis and show poor responses to immune checkpoint (IC) inhibitor (ICI) therapy. Competing endogenous RNA (ceRNA) networks, tumor-infiltrating immune cells (TIICs), and ICIs may influence tumor prognosis and response rates to ICI therapy. This study is aimed at identifying prognostic and IC-related biomarkers and key TIIC signatures to improve prognosis and ICI therapy response in HNSCC patients. Methods and Results Ninety-five long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and 1746 mRNAs were identified using three independent methods. We constructed a ceRNA network and estimated the proportions of 22 immune cell subtypes. Ten ceRNAs were related to prognosis according to Kaplan–Meier analysis. Two risk signatures based, respectively, on nine ceRNAs (ANLN, CFL2, ITGA5, KDELC1, KIF23, NFIA, PTX3, RELT, and TMC7) and three immune cell types (naïve B cells, neutrophils, and regulatory T cells) via univariate Cox regression, least absolute shrinkage and selection operator, and multivariate Cox regression analyses could accurately and independently predict the prognosis of HNSCC patients. Key mRNAs in the ceRNA network were significantly correlated with naïve B cells and regulatory T cells and with stage, grade, and immune and molecular subtype. Eight IC genes exhibited higher expression in tumor tissues and were correlated with eight key mRNAs in the ceRNA network in HNSCC patients with different HPV statuses according to coexpression and TIMER 2.0 analyses. Most drugs were effective in association with expression of these key signatures (ANLN, CFL2, ITGA5, KIF23, NFIA, PTX3, RELT, and TMC7) based on GSCALite analysis. The prognostic value of key biomarkers and associations between key ceRNAs and IC genes were validated using online databases. Eight key ceRNAs were confirmed to predict response to ICI in other cancers based on TIDE analysis. Conclusions We constructed two risk signatures to accurately predict prognosis in HNSCC. Key IC-related signatures may be associated with response to ICI therapy. Combinations of ICIs with inhibitors of eight key mRNAs may improve survival outcomes of HNSCC patients.

Differentiating Acute Interstitial Nephritis From Immune Checkpoint Inhibitors From Other Causes

2661 Background: Immune-related adverse effects (irAE) are autoimmune-like toxicities caused by immune checkpoint inhibitor (ICI) treatment and often necessitate interventions such as corticosteroids, treatment interruptions/discontinuation, or hospital admission. Although ICI related irAEs are well described in literature, data on the toxicity profile associated with long-term ICI use remains limited. Since the optimal duration of therapy with ICI agents is currently unknown, it is crucial to assess the risks of long-term ICI use. Methods: This was a retrospective, observational, single-center study of adult oncology patients who received at least 1 year of programed death 1 (PD-1) inhibitor or programmed death ligand-1 (PD-L1) inhibitor treatment. The objective of this study was to characterize late-onset irAEs defined as greater than 1 year with long-term ICI treatment. Clinically significant irAEs were defined as those requiring corticosteroid treatment, hospital admission, treatment interruption or treatment discontinuation. This study included patients who received at least 1 year of nivolumab, pembrolizumab, atezolizumab or durvalumab between January 2016 and September 2021. Disease states included head and neck cancer, renal cell carcinoma, non-small cell lung cancer, and melanoma. Patients with concurrent exposure to other treatment such as chemotherapy, radiation, surgery, tyrosine kinase inhibitors and monoclonal antibodies while on ICI treatment were included in the study. Exclusion criteria included patients with treatment breaks of greater than 6 months, two malignancies undergoing active treatment, and treatment administration outside of the study center. Results: Of 282 patients assessed, 143 met study inclusion criteria. The median ICI treatment duration was 19 months (IQR 14-27). There was a 30% incidence of late-onset irAEs, of which 22% were clinically significant. Most late-onset irAEs were low in severity, as 45 (90%) were grade 1-2 and 5 (10%) were grade 3. The most common late-onset irAEs were pulmonary (8%) and gastrointestinal (7%). Univariate analysis suggests risk factors potentially associated with late-onset irAEs include concurrent exposure to additional therapies during ICI treatment and past medical history of rheumatologic disease. Conclusions: Although the optimal duration of ICI therapy is unknown, this study suggests that long-term ICI use was associated with a low but notable incidence of toxicities, of which most were low in severity. [Table: see text]

BackgroundCancerous cells can utilize immune checkpoints to escape T-cell-mediated cytotoxicity. Agents that target PD-1, PD-L1 and CTLA4 are collectively deemed immune checkpoint inhibitors (ICIs), and many have been approved for...

Tissue Factor (TF) Is Upregulated in Tumor-Bearing Mice Treated with Immune Checkpoint Inhibitor (ICI)