Upregulation Of PD-1 Research Articles

Injury-induced myosin-specific tissue-resident memory T cells drive immune checkpoint inhibitor myocarditis

Cardiac myosin-specific (MyHC) T cells drive the disease pathogenesis of immune checkpoint inhibitor-associated myocarditis (ICI-myocarditis). To determine whether MyHC T cells are tissue-resident memory T (TRM) cells, we characterized cardiac TRM cells in naive mice and established that they have a distinct phenotypic and transcriptional profile that can be defined by their upregulation of CD69, PD-1, and CXCR6. We then investigated the effects of cardiac injury through a modified experimental autoimmune myocarditis mouse model and an ischemia-reperfusion injury mouse model and determined that cardiac inflammation induces the recruitment of autoreactive MyHC TRM cells, which coexpress PD-1 and CD69. To investigate whether the recruited MyHC TRM cells could increase susceptibility to ICI-myocarditis, we developed a two-hit ICI-myocarditis mouse model where cardiac injury was induced, mice were allowed to recover, and then were treated with anti-PD-1 antibodies. We determined that mice who recover from cardiac injury are more susceptible to ICI-myocarditis development. We found that murine and human TRM cells share a similar location in the heart and aggregate along the perimyocardium. We phenotyped cells obtained from pericardial fluid from patients diagnosed with dilated cardiomyopathy and ischemic cardiomyopathy and established that pericardial T cells are predominantly CD69+ TRM cells that up-regulate PD-1. Finally, we determined that human pericardial macrophages produce IL-15, which supports and maintains pericardial TRM cells.

Differential T-cell profiles determined by Hepatitis B surface antigen decrease among people with Human Immunodeficiency Virus /Hepatitis B Virus coinfection on treatment

BackgroundSeveral studies have reported that combination antiretroviral therapy (cART) enhances the hepatitis B surface antigen (HBsAg) clearance rate in Human Immunodeficiency Virus-1/Hepatitis B Virus (HIV/HBV) coinfected patients, yet the associated immunological characteristics remain unclear.MethodsGlobal and specific immune phenotypic profiles were examined in 48 patients with HIV/HBV coinfection before cART and at 1-year, and 3-year after cART using flow cytometry. In addition, 61 patients with HBV monoinfection were included for comparison.ResultsHBsAg response (sAg-R) was defined as > 0.5 log decrease within six months of cART initiation, and 16 patients achieved it. Patients with sAg-R (the sAg-R group) exhibited distinct immune phenotypes compared to those of HBsAg-retained patients (the sAg-NR group). Notably, patients with sAg-R had lower CD4+ T cell counts and a higher number of HBcAg-specific T cells. Further, the sAg-R group exhibited upregulation of HLA-DR, Ki67, and PD-1 in CD4+ T cells and heightened HLA-DR and T-bet in CD8+ T cells. However, the sAg-R group had fewer TEMRA cells but more TEM and Th17 cells than those in the sAg-NR group. Expression of various markers, including HLA-DR+CD4+, Ki67+CD4+, PD-1+CD4+, CD38+CD8+, HLA-DR+CD8+, TIM-3+CD8+, HBV-specific CD4+ T cell secreting IFN-γ and IL-2, and specific CD8+ T cell secreting IFN-γ and IL-2, correlated with HBsAg decrease.ConclusionThe decline in HBsAg levels during cART in HIV/HBV coinfection involves significant alterations in CD4+ and CD8+ T cells phenotypes, offering a novel perspective on a functional HBV cure.

Integrated proteomic and glycoproteomic analysis reveals heterogeneity and molecular signatures of brain metastases from lung adenocarcinomas

Brain metastasis is a major cause of poor prognosis and death in lung adenocarcinoma (LUAD); however, the understanding of therapeutic strategies and mechanisms for brain metastases from LUAD (BM-LUAD) remains notably limited, especially at the proteomics levels. To address this issue, we conducted integrated proteomic and glycoproteomic analyses on 49 BM-LUAD tumors, revealing two distinct subtypes of the disease: BM-S1 and BM-S2. Whole exome sequencing analysis revealed that somatic mutations in STK11 and KEAP1, as well as copy number deletions on chr19p13.3, such as STK11, UQCR11, and SLC25A23, were more frequently detected in BM-S2. In BM-S1 tumors, we observed significant infiltration of GFAP + astrocytes, as evidenced by elevated levels of GFAP, GABRA2, GABRG1 and GAP43 proteins and an enrichment of astrocytic signatures in both our proteomic data and external spatial transcriptomic data. Conversely, BM-S2 tumors demonstrated higher levels of PD-1 immune cell infiltration, supported by the upregulation of PD-1 and LAG-3 genes. These findings suggest distinct microenvironmental adaptations required by the different BM-LUAD subtypes. Additionally, we observed unique glycosylation patterns between the subtypes, with increased fucosylation in BM-S1 and enhanced sialylation in BM-S2, primarily affected by glycosylation enzymes such as FUT9, B4GALT1, and ST6GAL1. Specifically, in BM-S2, these sialylation modifications are predominantly localized to the lysosomes, underscoring the critical role of N-glycosylation in the tumor progression of BM-LUAD. Overall, our study not only provides a comprehensive multi-omic data resource but also offers valuable biological insights into BM-LUAD, highlighting potential mechanisms and therapeutic targets for further investigation.

Histopathologic, Molecular, and Clinical Profiling of Lymphoepithelioma-like Carcinoma of the Bladder

Lymphoepithelioma-like carcinoma of the bladder (LELC-B) is a rare histologic subtype characterized by strong immune cell (IC) infiltrates. A better prognosis and favorable response rates to immune checkpoint inhibitors have been described. We aimed to characterize the molecular profiles and IC infiltration of LELC-B for a better understanding of its therapeutic implications. We identified 11 muscle-invasive bladder cancer cases with pure and mixed LELC-B. Programmed cell death ligand-1 (PD-L1) expression and mismatch repair proteins were evaluated using immunohistochemistry. We calculated the tumor mutational burden and characterized mutational profiles using whole-exome DNA sequencing data. Transcriptomic signatures were detected using the NanoString nCounter PanCancer IO360 Panel. Multiplex immunofluorescence of tumor microenvironment (PD-L1, PanCK, α-SMA, vimentin, CD45, and Ki67) and T cells (CD4, CD3, PD-1, CD163, CD8, and FoxP3) was used to quantify cell populations. All LELC-B cases were highly positive for PD-L1 (median tumor proportion score/tumor cell, 70%; range, 20%-100%; median combined positive score, 100; range, 50-100) and mismatch repair proficient and negative for Epstein-Barr virus infection. IC infiltrates were characterized by a high CD8+ T-cell count and high PD-1/PD-L1 expression on immune and tumor cells. LELC-B showed upregulation of signaling pathways involved in IC response. Most common mutations were found in chromatin remodeling genes causing epigenetic dysregulation. All LELC-B cases showed high tumor mutational burden with a median of 39 mutations/Mb (IQR, 29-66 mutations/Mb). In conclusion, LELC-B is a highly immunogenic tumor, showing strong upregulation of PD-1/PD-L1 and making immune checkpoint inhibitors a promising treatment option.

Macrophages dig into the obesity paradox in cancer

Response to immune checkpoint blockade is increased in obesity-related cancers, but the mechanisms remain unclear. In a recent issue of Nature, Bader etal. report that obesity in mice induces macrophage PD-1 upregulation to promote tumor growth while potentiating immunotherapy responses.

Combination Atezolizumab, Cobimetinib, and Vemurafenib as a Treatment Option in BRAF V600 Mutation-Positive Melanoma: Patient Selection and Perspectives.

The treatment landscape for advanced and metastatic melanoma has drastically changed in recent years, with the advent of novel therapeutic options such as immune checkpoint inhibitors and targeted therapies offering remarkable efficacy and significantly improved patient outcomes compared to traditional approaches. Approximately 50% of melanomas harbor activating BRAF mutations, with over 90% resulting in BRAF V600E. Tumors treated with BRAF inhibitor monotherapy have a high rate of developing resistance within six months. Combination therapy with MEK inhibitors helped to mitigate this treatment resistance and led to improved outcomes. Due to the up-regulation of PD-1/PD-L1 receptors in tumors treated with BRAF/MEK inhibitor therapy, further studies included a third combination agent, anti-PD-1/PD-L1 inhibitors. This triple combination therapy may have superior efficacy and a manageable safety profile when compared with single or double agent therapy regimens.

Integrating system biology and intratumor gene therapy by trans-complementing the appropriate co-stimulatory molecule as payload in oncolytic herpes virus

Systems biology has been applied at the multi-scale level within the cancer field, improving cancer prevention, diagnosis and enabling precision medicine approaches. While systems biology can expand the knowledge and skills for oncological treatment, it also represents a challenging expedition due to cancer complexity, heterogeneity and diversity not only between different cancer indications, but also in its evolution process through space and time. Here, by characterizing the transcriptional perturbations of the tumor microenvironment induced by oncolytic, we aimed to rationally design a novel armed oncolytic herpes virus. We found that intratumor oncovirotherapy with HSV-1 induces T-cell activation signatures and transcriptionally activates several costimulatory molecules. We identified differentially expressed costimulatory receptors and binding partners, where inducible co-stimulators (ICOS) resulted in the potentially most beneficial targeted therapy. Through an ex-vivo transcriptomic analysis, we explored the potential of arming an oncolytic virus as a combination therapy strategy; in particular, we engineered a targeted herpes virus encoding ICOSL (THV_ICOSL), which resulted in a significant improvement in tumor size control compared to unarmed parental virus. Also, combination with a PD-1 inhibitor enhanced antitumor efficacy as predictable by upregulation of PD-1 and ligands pair (PD-L1/PD-L2) upon oncolytic virus injection. Generation of the human version of this virus encoding hICOSL orthologue effectively and specifically activated human T cells by triggering the ICOS pathway. Our data support the data-driven generation of armed oncolytic viruses as combination immunotherapeutic with checkpoint inhibitors.

EVICTION study: ICT01, an anti-Butyrophilin 3A monoclonal antibody activating γ9δ2 T cells in combination with pembrolizumab in checkpoint inhibitor refractory melanoma.

9534 Background: γ9δ2 T-cell tumor infiltration is associated with a favorable prognosis making these cells a new potential target for immunotherapy. The anti-BTN3A mAb ICT01 selectively activates γ9δ2 T cells and is being studied in the phase1/2a EVICTION Trial (NCT04243499). In preclinical studies, ICT01 induces upregulation of PD-1 on γ9δ2 T-cells and combination with pembrolizumab leads to enhanced cancer cell killing, providing scientific rationale to evaluate this combination. Here we present interim results from EVICTION of ICT01 in combination with pembrolizumab in patients with checkpoint inhibitor (CPI) refractory melanoma. Methods: EVICTION evaluated ICT01 (20 µg to 200 mg, Q3W) plus pembrolizumab (200mg IV Q3W) in a dose escalation solid tumor basket design that resulted in an ongoing expansion cohort of patients with CPI refractory melanoma (2 dose levels, 7 and 200 mg ICT01). Patients are selected based on higher baseline γ9δ2 T cells. Efficacy evaluations by i/RECIST 1.1 are conducted every 8 weeks. Disease Control Rate (DCR) as primary efficacy endpoint is defined as the sum of complete response (CR), partial response (PR) and stable disease (SD, minimum week 16). Baseline and on-treatment blood and biopsy specimens are collected for correlative translational work. Results: ICT01 plus pembrolizumab has a favorable safety profile with first-dose Grade 1/2 infusion related reactions (IRR) and cytokine release syndrome (CRS) as most common adverse events (in 38% and 19% of patients respectively, 5%and 2% Grade 3) among all doses and indications. To date, 35 CPI refractory melanoma patients have been enrolled, of which 65% have received 2+ prior lines of CPI. Currently 21 patients are evaluable at week 16 with a DCR of 42% (including 3 PR) and a 6-month progression free survival of 38%. In the circulation, full BTN3A receptor occupancy on immune cells was achieved at 7 mg dose, with down-modulation of BTN3A observed at doses greater than 20 mg. Clinical response was related to baseline tumoral BTN3A expression, sustained elevation of IFNg levels, and tumor microenvironment remodeling, including increased PD-L1 expression and CD8 T cells proliferation and activation. Conclusions: ICT01 in combination with pembrolizumab has a favorable safety profile and promising efficacy data. Patient selection based on BTN3A tumor expression will be further evaluated as an enrichment strategy. Clinical trial information: NCT04243499 .

Upregulation of PD-1 and its ligands and expansion of T peripheral helper cells in the nephritic kidneys of lupus-prone BXSB-Yaa mice.

This study aimed to investigate the role of the programmed cell death protein 1 (PD-1) pathway and T peripheral helper (Tph) cells in the pathogenesis of lupus nephritis using lupus-prone BXSB-Yaa mice. Male BXSB-Yaa mice and age-matched male C57BL/6 mice were used. The expression of PD-1 and its ligands (programmed cell death 1 ligand-1, PD-L1 and programmed cell death 1 ligand-2, PD-L2) and the phenotypes of kidney-derived cells and splenocytes expressing these molecules were analyzed by immunofluorescence and flow cytometry. Nephritis spontaneously developed in 16-week-old but not in 8-week-old BXSB-Yaa or C57BL/6 mice. PD-1 was expressed on CD4+ mononuclear cells (MNCs) that infiltrated the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of CD4+PD-1+CXCR5-ICOS+ kidney-derived Tph cells was higher in 16-week-old than in 8-week-old BXSB-Yaa and C57BL/6 mice, whereas the frequency of CD4+PD-1+CXCR5+ICOS+ kidney-derived T follicular helper (Tfh) cells was not significantly different between the mice. PD-L1 was constitutively expressed in the renal tubules. PD-L2 was expressed in the glomeruli of 16-week-old BXSB-Yaa mice. The frequency of PD-L1highCD11c+CD3-CD19- and PD-L2+CD11c+CD3-CD19- kidney-derived MNCs in 16-week-old BXSB-Yaa mice was significantly higher than that of the control mice. The percentage of kidney-derived Tph cells but not Tfh cells was correlated with the urinary protein levels in the nephritic mice. The results of this study suggest that kidney-infiltrating PD-1+ Tph cells expanded concomitantly with the upregulation of PD-L1 and PD-L2 in the kidneys and the progression of lupus nephritis.

Abstract 7632: Final survival outcomes and post-hoc tumor gene expression pathway analyses of complete responders from a phase Ib clinical trial of HSP90 inhibitor onalespib and paclitaxel in patients with advanced triple-negative breast cancer

Abstract Background: Heat shock protein 90 (HSP90) is a molecular chaperone required for stabilization of client proteins over-activated in triple-negative breast cancer (TNBC). Over-expression of HSP90 client proteins has been implicated in paclitaxel resistance, therefore, it was hypothesized that the potent HSP90 inhibitor onalespib could improve paclitaxel efficacy. As TNBCs are a heterogenous population of tumors, it is important to establish biomarkers of response to this treatment combination. Methods: Patients with TNBC were treated with intravenous (IV) paclitaxel and IV onalespib at doses ranging from 120 mg/m2 to 260 mg/m2 on days 1, 8, and 15 of a 28-day cycle utilizing a standard 3+3 design. The primary objectives were to determine dose-limiting toxicities and maximum tolerated dose. Secondary objectives included determination of overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS). Retrospectively, archival tumor tissue was obtained from study patients and processed for RNA expression analysis by NanoString nCounter platform with the Breast Cancer 360 panel. Post-hoc gene expression analysis was performed through Qiagen Ingenuity Pathway Analysis (IPA). Linear models were employed to detect differential expression between conditions while considering cartridge effect. Results: 31 patients were enrolled in this study, of which 90% received prior taxane therapy. The established recommended phase II dose was 260 mg/m2 onalespib when given with 80 mg/m2 paclitaxel. ORR was 20%, median DOR was 5.6 months. Three patients achieved complete responses (CR), all of whom had received prior taxane therapy. Nanostring profiling analysis revealed that patients who had a CR to treatment expressed higher levels of several breast cancer-associated genes also known to be dependent upon HSP90 activity compared to patients with progressive disease (PD) as their best treatment response. These genes include FGFR4, JAK2, PIK3R1, TGFB3, and TGFBR2 (all p<0.05). Tumor genes overexpressed in patients with PD versus CR included IRF6, MMP7, and KRT17 (all p<0.01). Additionally, IPA showed patients with CR to have substantial upregulation of PD-1, PD-L1, CTLA-4, p53, HER2, and cyclin/cell cycle regulatory signaling pathways compared to patients with PD (all p<0.01). Conclusions: Combination treatment with onalespib and paclitaxel had an acceptable toxicity profile and showed anti-tumor activity in patients with advanced TNBC. Gene expression analysis of patient tumor samples suggest that TNBCs with greater activation of immune checkpoint pathways and those with proteins dependent on HSP90 activity, including p53 and HER2, may be more susceptible to HSP90 inhibition. ClinicalTrials.gov study identification: NCT02474173 Citation Format: Dionisia Marie Quiroga, Himanshu Savardekar, Emily Schwarz, Nicole O. Williams, Courtney Johnson, Adam Brufsky, Mara Chambers, Saveri Bhattacharya, Maria Patterson, Sagar D. Sardesai, Daniel Stover, Maryam Lustberg, Anne Noonan, Mathew Cherian, Darlene M. Bystry, Kasey Hill, Min Chen, Mitch A. Phelps, Julie Stephens, Lianbo Yu, Bhuvaneswari Ramaswamy, Robert Wesolowski, William E. Carson. Final survival outcomes and post-hoc tumor gene expression pathway analyses of complete responders from a phase Ib clinical trial of HSP90 inhibitor onalespib and paclitaxel in patients with advanced triple-negative breast cancer [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 7632.

Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Post-translational Modification of PD-1: Potential Targets for Cancer Immunotherapy.

Activation of effector T cells leads to upregulation of PD-1, which can inhibit T-cell activity following engagement with its ligand PD-L1. Post-translational modifications (PTM), including glycosylation, phosphorylation, ubiquitination, and palmitoylation, play a significant role in regulating PD-1 protein stability, localization, and interprotein interactions. Targeting PTM of PD-1 in T cells has emerged as a potential strategy to overcome PD-1-mediated immunosuppression in cancer and enhances antitumor immunity. The regulatory signaling pathways that induce PTM of PD-1 can be suppressed with small-molecule inhibitors, and mAbs can directly target PD-1 PTMs. Preliminary outcomes from exploratory studies suggest that focusing on the PTM of PD-1 has strong therapeutic potential and can enhance the response to anti-PD-1.

Upregulation of PD-1/PD-L1 and downregulation of immune signaling pathways lead to more severe visceral leishmaniasis in undernutrition mice

BackgroundLeishmaniasis is mainly prevalent in tropical and subtropical developing countries, where chronic undernutrition often co-exists. Undernutrition is reported to promote the progression of leishmaniasis, but its immune mechanisms have not been fully elucidated.MethodsTo simulate chronic undernutrition of patients in epidemic areas and explore the immune mechanism of undernutrition promoting leishmaniasis, BALB/c mouse models with different nutritional imbalances were established, including undernutrition 75%, undernutrition 65% and obesity mouse models. After infection with Leishmania donovani in these model mice, we focused on evaluating the progress of leishmaniasis in the spleen and liver, the expression of important immunosuppressive and immunoactivation molecules, and changes of spleen transcriptome. The immune signaling pathways enriched by differentially expressed genes and hub genes were analyzed.ResultsThe results showed that among the mouse infection models, undernutrition 75% + infection group had the highest parasite load in the spleen and liver at the 8th week post-infection, possibly due to the continuous increase of PD-1, PD-L1 and TCR. Spleen RNA-seq results suggested that some immune signaling pathways were downregulated in undernutrition 75% + infection group, including neutrophil extracellular trap formation, IL-17 signaling pathway, natural killer cell-mediated cytotoxicity, etc. Among them, neutrophil extracellular trap formation pathway had the largest number of downregulated genes. This also explained why undernutrition 75% + infection group had the highest parasite load. Through PPI network analysis, hub genes such as Lcn2, Ltf, Mpo, Dnaja1, Hspa1a, Hspa1b and Hsph1 were screened out and might play important roles in the process of undernutrition promoting leishmaniasis.ConclusionsUndernutrition upregulated PD-1 and PD-L1 expression and downregulated immune signaling pathways in mice with visceral leishmaniasis. The signaling pathways and hub genes may serve as drug targets or intervention targets for the treatment of leishmaniasis patients with undernutrition.Graphical

PD-1 signaling in neonates restrains CD8+ T cell function and protects against respiratory viral immunopathology

Respiratory viral infections, including human metapneumovirus (HMPV), remain a leading cause of morbidity and mortality in neonates and infants. However, the mechanisms behind the increased sensitivity to those respiratory viral infections in neonates are poorly understood. Neonates, unlike adults, have several anti-inflammatory mechanisms in the lung, including elevated baseline expression of programmed death ligand 1 (PD-L1), a ligand for the inhibitory receptor programmed cell death protein 1 (PD-1). We thus hypothesized that neonates would rely on PD-1:PD-L1 signaling to restrain antiviral CD8 responses. To test this, we developed a neonatal primary HMPV infection model using wild-type C57BL/6 (B6) and Pdcd1-/- (lacking PD-1) mice. HMPV-infected neonatal mice had increased PD-L1/PD-L2 co-expression on innate immune cells but a similar number of antigen-specific CD8+ T cells and upregulation of PD-1 to that of adult B6 mice. Neonatal CD8+ T cells had reduced interferon-gamma (IFN-γ), granzyme B, and interleukin-2 production compared with B6 adults. Pdcd1-/- neonatal CD8+ T cells had markedly increased production of IFN-γ and granzyme B compared with B6 neonates. Pdcd1-/- neonates had increased acute pathology with HMPV or influenza. Pdcd1-/- neonates infected with HMPV had long-term changes in pulmonary physiology with evidence of immunopathology and a persistent CD8+ T-cell response with increased granzyme B production. Using single-cell ribonucleic acid sequencing from a child lacking PD-1 signaling, a similar activated CD8+ T-cell signature with increased granzyme B expression was observed. These data indicate that PD-1 signaling critically limits CD8+ T-cell effector functions and prevents immunopathology in response to neonatal respiratory viral infections.

Toll-like Receptor-9 Agonist Reverses T Cell Exhaustion in a Murine Model of Pediatric B-ALL

Despite significant treatment advances, the poor long-term prognosis for children with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) persists. The application of immune cell-based therapies, such as chimeric antigen receptor-expressing T cells and bispecific T-cell engagers, has improved progression-free survival for many B-ALL patients. However, the majority of these patients still experience disease relapse. An improved understanding of the mechanisms that impair T cell function in B-ALL patients could inform strategies to further improve outcomes. Recent studies have revealed T cell exhaustion, especially as indicated by PD-1 and Tim- 3 expression, to be predictive of disease relapse and overall survival in B-ALL. However, the precise contribution of T cell dysfunction to B-ALL development remains unclear. In this study, we explore the biology that leads to impaired T cell function and investigate potential approaches to restore it using both transgene-driven and syngeneic leukemia cell transplant models of B-ALL. First, we used the Emu-Ret transgenic mouse model of hyperdiploid B-ALL to investigate the induction of exhaustion-associated marker expression (PD-1, CTLA-4 and Tim-3) on CD4 and CD8 T cells. Longitudinal analysis of marker expression in various settings revealed that an upregulation of PD-1 and Tim-3, but not CTLA-4, on T cells occurred only in the presence of leukemia cells, not during the extended, nascent preleukemia phase. This suggests that T cell exhaustion occurs exclusively during the established malignancy phase, highlighting the dynamic nature of T cell responses as disease progresses. Next, B-ALL cells from moribund Emu-Ret mice were transplanted into 4-6-week-old wild type BALB/c mice and markers of T cell exhaustion were measured as disease progressed. Expression of PD-1 and Tim-3 on T cells correlated with disease progression. Notably, a significant increase in these markers was already detectable at low leukemia burden. These results indicate that T cell exhaustion is not simply a reflection of high disease burden and confirm the differing abilities of preleukemic and leukemic cells to affect T cell phenotype. Previously, we have demonstrated the ability of toll-like receptor (TLR) ligands to induce immune control over B-ALL cells. Thus, we investigated the impact of CpG oligodeoxynucleotides (CpG ODN), a ligand for TLR-9, on B-ALL depletion and T cell dysfunction. The treatment of leukemia bearing mice with CpG ODN led to a decrease in PD-1 and Tim-3 expression on T cells, which was also associated with a reduction in leukemia burden. We are currently investigating the functional implications of these expression changes using in vitro assays of T cell proliferation, cytokine production, and cytotoxicity. Additionally, we are also assessing if anti-tumour control by T cells can be revived in vivo, by comparing B-ALL progression and mouse survival after administration of antibodies against exhaustion markers, with or without CpG ODNs as adjuvant therapy. Overall, this study reveals that the functional impact of interactions between B-ALL cells and the immune system evolves during leukemia development. These insights could inform therapeutic strategies to overcome B-ALL immune evasion mechanisms and enhance patient outcomes.

Beyond Mitigating Immune-Related Adverse Events: Glucocorticoids Ameliorate Bispecific Antibody-Mediated T-Cell Exhaustion I n Vitro

Following the approval of blinatumomab in 2015 T-cell recruiting bispecific antibodies (BsAbs) became the established standard-of-care for relapsed or refractory (r/r) and minimal residual disease positive B-cell precursor acute lymphoblastic leukemia. More recently, there has been a notable increase in the approval of additional BsAb constructs for malignancies such as r/r follicular lymphoma, r/r large B-cell lymphoma, and advanced multiple myeloma (MM), with further approvals anticipated. Despite the clinical success, administration of BsAbs is commonly linked to dose-limiting immune-related adverse events (IrAEs), such as cytokine release syndrome, possibly compromising treatment efficacy. Hence, current BsAb as well as CAR T approvals integrate prophylactic and acute glucocorticoid (GC) application upon occurrence of IrAEs. Surprisingly, despite their immunosuppressive nature, GC administration had no negative impact on response or survival of MM patients receiving CAR T-cell therapy (Wang 2022). Based on these data, we hypothesize that the GC-mediated downregulation of BsAb-induced T-cell function has a dual effect: mitigating IrAEs and ameliorating T-cell exhaustion. Here we tested the effect of simultaneous administration of the GC dexamethasone (dexa) and a CD3xCD19 targeting BsAb on T-cell function and exhaustion. We first evaluated the short-term effect of dexa exposure on BsAb-mediated cytotoxicity by co-culturing healthy donor (HD) T cells with CD19 + cell lines and stimulating with a CD3xCD19 BsAb alone or in combination with 300nM dexa for 3-5 days. In fact, the addition of dexa decreased BsAb-mediated tumor cell lysis against OCI-Ly1 cells after 3 days, but not after 5 days. Surprisingly, however, T-cells showed a significantly increased expansion upon dexa exposure after 3 and 5 days (day 3: Ki-67 MFI ratio BsAb=337.2, +dexa=676.0, p=0.008; day 5: CD2 +-fold change to day 0 BsAb only=7.1; +dexa=11.6; p=0.01). In cytotoxicity assays against murine Ba/F3 cells transduced with human CD19 with or without the co-stimulatory molecule CD86, T cells showed significantly reduced BsAb-mediated cytotoxicity and proliferation upon dexa exposure in the absence of CD86. Intriguingly, however, T-cell function was rescued upon CD86 co-expression. To evaluate long-term effects of GCs on T-cell function and exhaustion, we next used our established long-term in vitro model system to mimic continuous stimulation of T cells with a BsAb ± GCs. HD T cells were co-cultured with CD19 + OCI-Ly1 cells for 28 days and continuously stimulated with a CD3xCD19 BsAb alone or in combination with dexa. T-cell fitness was assessed longitudinally. Surprisingly, but in line with our short-term data, we found increased CD8 + T-cell expansion upon BsAb +dexa co-exposure in long-term cultures (abs. CD8 + cell count day 21 BsAb=1.613, +dexa=34.583). Furthermore, immunophenotyping revealed similar upregulation of the checkpoint inhibitors PD-1, Tim-3, Lag-3 after 7 days. On day 21, however, Lag-3 expression on dexa exposed CD4 + and CD8 + T cells was decreased (MFI ratio BsAb=10.5, +dexa=1.7, p=0.02). Inflammatory cytokine (IL-2, IFN-γ, TNF-α) levels were decreased in the supernatant of dexa-exposed vs BsAb only co-cultures. However, low levels of IFN-γ were maintained in the dexa co-exposed condition over 24 days (mean level IFN-γ day 24 BsAb=53pg/ml, +dexa=1756 pg/ml). Finally, T cells were isolated from cultures every 7 days and tested in 72h cytotoxicity assays, without further exposure to dexa. Intriguingly, T cells previously exposed to dexa maintained a significantly higher cytotoxicity over 28 days (mean % specific lysis day 21 BsAb=34.2, +dexa=90.3, p=0.004). Taken together, our data demonstrate a positive impact of GCs on BsAb-mediated T-cell expansion in short- and long-term setting. Characterization of T cells upon long term BsAb exposure demonstrated an ameliorating effect of GCs on BsAb-induced T-cell exhaustion. Our data suggest that dampening of T-cell activity by GC administration in BsAb-treated patients could serve two purposes: managing IrAEs while simultaneously ameliorating T-cell exhaustion and improving T-cell persistence. In future studies we will further dissect the effect of dexa on the transcriptional landscape of BsAb-stimulated T cells by performing RNA sequencing and also explore the effect of GCs on CAR T-cell function.

Lineage-specific regulation of PD-1 expression in early-stage hepatocellular carcinoma following 90yttrium transarterial radioembolization – Implications in treatment outcomes

BackgroundHepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related deaths in the world. Liver-directed therapies, including 90Yttrium (90Y) radioembolization, play an integral role in the management of HCC with excellent response rates. This has led to clinical trials of immunotherapy in combination with 90Y. Elevated PD-1 expression and lymphopenia were recently shown as risk factors for disease progression in early-stage HCC treated with liver-directed therapies. The aim of this study was to investigate PD-1 expression dynamics in bridge/downstage to transplant in HCC patients receiving first-cycle 90Y and evaluate the impact of these changes on response rates and time-to-progression (TTP). MethodsPatients with HCC receiving first-cycle 90Y as a bridge to liver transplantation (n = 99) were prospectively enrolled. Blood specimens were collected before 90Y and again during routine imagining follow-up to analyze PD-1 expression via flow cytometry. Complete and objective response rates (CR and ORR) were determined using mRECIST. ResultsIn 84/88 patients with available follow-up imaging, 83% had a localized ORR with 63% having localized CR. For overall response, 71% and 54% experienced ORR and CR, respectively. Post-90Y PD-1 upregulation in CD8 + associated with HCC progression and decreased TTP. Treatment with 90Y was associated with an anticipated significant post-treatment drop in lymphocytes (P < 0.001) that was independent of PD-1 expression for either CD4+ or CD8+ T cells (P = 0.751 and P = 0.375) and not associated with TTP risk. The change in lymphocytes was not correlated with PD-1 expression following treatment nor TTP. ConclusionsElevated PD-1 expression on peripheral T cells is associated with increased risk of HCC progression and shorter time to progression in bridging/downstaging to transplant HCC patients undergoing first-cycle 90Y. Treatment-induced lymphopenia was not associated with treatment response, or increased progression risk, suggesting this anticipated adverse event does not impact short-term HCC outcomes.

Unraveling the genetic associations between PD-1/PD-L1 and 13 circulating biomarkers linked to physiological and pathological processes.

Evidence of the genetic interconnectedness between PD-1/PD-L1 and circulating biomarkers related to physiological and pathological processes is largely unclear. Understanding these genetic links is crucial for gaining insights into the underlying mechanisms and potential implications in cancer immunotherapy. To shed light on potential roles of 90 circulating biomarkers in PD-1/PD-L1, we conducted a comprehensive Mendelian randomization (MR) analysis, leveraging genetic data from large-scale genome-wide association studies. Our results revealed negative associations between EN-RAGE and TRAIL-R2 with PD-1 levels. Additionally, we observed that PD-1 levels were positively associated with TRAIL, VEGF, and ANPEP, indicating their potential role in PD-1 upregulation. Furthermore, our analysis revealed causal associations between several circulating proteins and PD-L1 levels. Thrombomodulin, PSGL-1, TNFSF14, renin, follistatin, β-NGF, KLK6, and MMP-7 demonstrated significant effects on PD-L1 regulation, suggesting their potential inhibitory role in immune checkpoint regulation. Eventually, we confirmed the potential roles of key genes involved in above circulating proteins in influencing the response to immunotherapy. Our findings provide valuable evidence of the genetic interconnectedness between PD-1/PD-L1 and circulating proteins related to physiological and pathological processes, shedding light on their potential roles in disease progression and therapeutic interventions.

The role of radiotherapy in tumor immunity and the potential of PET/CT in detecting the expression of PD-1/PD-L1.

Upregulation of PD-1/PD-L1 allows cancer cells to escape from host immune systems by functionally inactivating T-cell immune surveillance. Clinical blockade strategies have resulted in an increased prevalence of patients with late-stage cancers. However, many cancer patients had limited or no response to current immunotherapeutic strategies. Therefore, how to improve the sensitivity of immunotherapy has become the focus of attention of many scholars. Radiotherapy plays a role in the recruitment of T cells in the tumor microenvironment, increases CD4 + and CD8 + T cells, and increases PD-L1 expression, resulting in the synergistically enhanced antitumor effect of irradiation and PD-L1 blockade. Radiotherapy can cause changes in tumor metabolism, especially glucose metabolism. Tumor glycolysis and tumor immune evasion are interdependent, glycolytic activity enhances PD-L1 expression on tumor cells and thus promotes anti-PD-L1 immunotherapy response. Therefore, the mechanism of radiotherapy affecting tumor immunity may be partly through intervention of tumor glucose metabolism. Furthermore, some authors had found that the uptake of 2'-deoxy-2'-[18F]fluoro-D-glucose(18F-FDG) was correlated with PD-1/PD-L1 expression. Positron emission tomography/computed tomography (PET/CT) is a non-invasive detection method for PD-1/PD-L1 expression and has several potential advantages over immunohistochemical (IHC), PET/CT can dynamically reflect the expression of PD-1/PD-L1 inside the tumor and further guide clinical treatment.