T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.

One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.

Transarterial Embolization Enhances Programmed Cell Death Ligand 1 Expression and Influences CD8+T Lymphocytes Cytotoxicity in an Orthotopic Hepatocellular Carcinoma Rat Model.

PURPOSE: To investigate the influence of transarterial embolization (TAE) on programmed cell death-ligand 1(PD-L1) expression and CD8+T tumour infiltrative lymphocyte cytotoxicity in the Sprague-Dawley (SD) rat model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: An orthotopic HCC model was established in twenty SD rats treated with TAE (lipiodol, n = 10) or sham (normal saline, n = 10) using homologous N1S1 hepatoma cells. Rats were euthanized 1 week after embolization. Flow cytometry was used to assess the proportion of CD4+T, CD8+T and programmed cell death-1+(PD-1+) CD8+T lymphocytes in the spleens and tumours. Distribution of CD8+T, granzyme-B+CD8+T lymphocytes and PD-L1+ cells was assessed by immunohistochemistry (IHC) or multiplex IHC. p value < 0.05 was considered statistically significant. RESULTS: The CD4/CD8 ratio and PD-1+CD8+ T lymphocytes exhibited higher values in TAE-treated tumours compared to sham-treated tumours (p = 0.021 and p = 0.071, respectively). Conversely, the number of CD8+T lymphocytes was decreased in TAE-treated tumours (p = 0.043), especially in the central region (p = 0.045). However, more CD8+T lymphocytes were found infiltrating the marginal region than central region in TAE-treated tumours (p = 0.046). The proportion of granzyme-B+CD8+T lymphocytes and the PD-L1 positive areas was elevated in tumours that treated with TAE (p all < 0.05). There was a negative correlation between PD-L1 expression and the number of infiltration of CD8+ T lymphocytes (p = 0.036). CONCLUSIONS: Immune cells are distributed unevenly in the tumours after TAE. The intrinsic induction state of the tumour after embolization may be insufficient to elicit a maximal response to PD-1/PD-L1 inhibitors.

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen.

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

The development of anti-PD-1 antibody-induced spinal cord injury in bone marrow transplant C57BL/6 Rag1-/- mouse model.

Aims: This paper was to scrutinize the toxicity mechanism of anti-programmed death 1 (anti-PD-1) therapy-caused spinal cord injury (SCI). Methods: Bone marrow transplant Rag1-/- mice were used to establish SCI model. Results: Anti-PD-1 results in SCI via CD8+ T-cells activation, while excessive activation of CD8+ T-cells further aggravated SCI. Both anti-PD-1 and the activation of CD8+ T-cells induced the expression of apoptosis-related perforin, GrB and FasL, but suppressed PI-9 level. The opposite results were observed in the effects of neuroserpin on these factors. CD8+ T-cells activation induced neurotoxicity via upregulation perforin, GrB and FasL and inhibiting PI-9. Additionally, neuroserpin suppressed CD8+ T-cells activation via perforin/GrB/PI-9/FasL pathways. Conclusion: These results may provide theoretical foundation for the clinical treatment of SCI caused by anti-PD-1.

What is this article about? In the process of treating cancer, immune checkpoint inhibitors such as anti-programmed death 1 (anti-PD-1) therapy, as a form of immunotherapy, have developed rapidly and changed the way to manage cancers significantly. However, some cancer patients who receive immune checkpoint blockade treatment suffer from severe adverse effects including spinal cord injury (SCI). This article for the first time constructed a bone marrow transplant mouse model to explore the toxicity mechanism of anti-PD-1 therapy-caused SCI.What were the results? We found that anti-PD-1 therapy can induce the activation of immune cells, while immune cell activation further promotes self-destruction of nerve cells by regulating cell death pathways.What do the results of the study mean? The mechanism of anti-PD-1 therapy-caused SCI is to activate of immune cells through regulating cell death pathways, thereby inducing self-destruction of nerve cells. These findings provide theoretical foundation for the clinical treatment of SCI caused by anti-PD-1 therapy.

B and T cells: (Still) the dominant orchestrators in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a severe hepatopathy characterized by hypergammaglobulinemia, presence of serum autoantibodies and histological appearance of interface hepatitis. Liver damage in AIH is initiated by the presentation of a liver autoantigen to uncommitted Th0 lymphocytes, followed by a cascade of effector immune responses culminating with the production of inflammatory cytokines, activation of cytotoxic cells and subsequent hepatocyte injury. B cells actively participate in AIH liver damage by presenting autoantigens to uncommitted T lymphocytes. B cells also undergo maturation into plasma cells that are responsible for production of immunoglobulin G and autoantibodies, which mediate antibody dependent cell cytotoxicity. Perpetuation of effector immunity with consequent progression of liver damage is permitted by impairment in regulatory T cells (Tregs), a lymphocyte subset central to the maintenance of immune homeostasis. Treg impairment in AIH is multifactorial, deriving from numerical decrease, reduced suppressive function, poor response to IL-2 and less stable phenotype. In this review, we discuss the role of B and T lymphocytes in the pathogenesis of AIH. Immunotherapeutic strategies that could limit inflammation and halt disease progression while reconstituting tolerance to liver autoantigens are also reviewed and discussed.

Impact of neoadjuvant chemotherapy on perioperative immune function in breast cancer patients: a propensity score-matched retrospective study.

To evaluate the impact of neoadjuvant chemotherapy on perioperative immune function in breast cancer patients, focusing on CD3+, CD4+, CD8+, and natural killer (NK) cells, as well as the CD4+/CD8+ ratio. We retrospectively reviewed medical records of breast cancer patients who underwent surgery with or without neoadjuvant chemotherapy at our medical center from January 2020 to December 2022. Patients were matched 1:1 based on propensity scores. Immune cell proportions and the CD4+/CD8+ ratio were compared on preoperative day one and postoperative days one and seven. Among matched patients, immune cell proportions and the CD4+/CD8+ ratio did not significantly differ between those who received neoadjuvant chemotherapy and those who did not at any of the three time points. Similar results were observed in chemotherapy-sensitive patients compared to the entire group of patients who did not receive neoadjuvant chemotherapy. However, chemotherapy-insensitive patients had significantly lower proportions of CD4+ and NK cells, as well as a lower CD4+/CD8+ ratio, at all three time points compared to patients who did not receive neoadjuvant chemotherapy. Neoadjuvant chemotherapy may impair immune function in chemotherapy-insensitive patients, but not in those who are sensitive to the treatment.

Unraveling clonal CD8 T cell expansion and identification of essential factors in γ-herpesvirus-induced lymphomagenesis.

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development.

LAG-3 and PD-1 synergize on CD8+ T cells to drive T cell exhaustion and hinder autocrine IFN-γ-dependent anti-tumor immunity.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

Blockade of LAG-3 and PD-1 leads to co-expression of cytotoxic and exhaustion gene modules in CD8+ T cells to promote antitumor immunity.

Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

Immunological Profiling of CD8+ and CD8- NK Cell Subpopulations and Immune Checkpoint Alterations in Early-Onset Preeclampsia and Healthy Pregnancy.

Despite the numerous studies on the clinical aspects of early-onset preeclampsia, our understanding of the immunological consequences of inadequate placenta development remains incomplete. The Th1-predominance characteristic of early-onset preeclampsia significantly impacts maternal immunotolerance, and the role of immune checkpoint molecules in these mechanisms is yet to be fully elucidated. Our study aims to fill these crucial knowledge gaps. A total of 34 pregnant women diagnosed with early-onset preeclampsia and 34 healthy pregnant women were enrolled in this study. A mononuclear cell fragment from the venous blood was separated and frozen. The CD8+ and CD8- NK cell subpopulations were identified and compared to their immune checkpoint molecule expressions using multicolor flow cytometry. The serum CD226 levels were measured by ELISA. Based on our measures, the frequency of the CD8- subpopulation was significantly higher than that of the CD8+ counterpart in both the NKdim and NKbright subsets. Significantly lower CD226 surface expressions were detected in the preeclamptic group compared to healthy women in all the investigated subpopulations. However, while no difference was observed in the level of the soluble CD226 molecule between the two groups, the CD112 and CD155 surface expressions were significantly different. Our study's findings underscore the significant role of the CD8+ and CD8- NK subpopulations in the Th1-dominated immune environment. This deepens our understanding of early-onset preeclampsia and suggests that each subpopulation could contribute to the compensation mechanisms and the restoration of the immunological balance in this condition, a crucial step toward developing effective interventions.

Epithelium/imcDC2 axis facilitates the resistance of neoadjuvant anti-PD-1 in human NSCLC.

BACKGROUND: Therapeutic resistance is a main obstacle to achieve long-term benefits from immune checkpoint inhibitors. The underlying mechanism of neoadjuvant anti-PD-1 resistance remains unclear. METHODS: Multi-omics analysis, including mass cytometry, single-cell RNA-seq, bulk RNA-seq, and polychromatic flow cytometry, was conducted using the resected tumor samples in a cohort of non-small cell lung cancer (NSCLC) patients received neoadjuvant anti-PD-1 therapy. Tumor and paired lung samples acquired from treatment-naïve patients were used as a control. In vitro experiments were conducted using primary cells isolated from fresh tissues and lung cancer cell lines. A Lewis-bearing mouse model was used in the in vivo experiment. RESULTS: The quantity, differentiation status, and clonal expansion of tissue-resident memory CD8+ T cells (CD8+ TRMs) are positively correlated with therapeutic efficacy of neoadjuvant anti-PD-1 therapy in human NSCLC. In contrast, the quantity of immature CD1c+ classical type 2 dendritic cells (imcDC2) and galectin-9+ cancer cells is negatively correlated with therapeutic efficacy. An epithelium/imDC2 suppressive axis that restrains the antitumor response of CD8+ TRMs via galectin-9/TIM-3 was uncovered. The expression level of CD8+ TRMs and galectin-9+ cancer cell-related genes predict the clinical outcome of anti-PD-1 neoadjuvant therapy in human NSCLC patients. Finally, blockade of TIM-3 and PD-1 could improve the survival of tumor-bearing mouse by promoting the antigen presentation of imcDC2 and CD8+ TRMs-mediated tumor-killing. CONCLUSION: Galectin-9 expressing tumor cells sustained the primary resistance of neoadjuvant anti-PD-1 therapy in NSCLC through galectin-9/TIM-3-mediated suppression of imcDC2 and CD8+ TRMs. Supplement of anti-TIM-3 could break the epithelium/imcDC2/CD8+ TRMs suppressive loop to overcome anti-PD-1 resistance. TRIAL REGISTRATION NUMBER: NCT03732664.

The relationship between CXCR6+CD8+T cells and clinicopathological parameters in patients with primary biliary cholangitis.

BACKGROUND: CXCR6+CD8+T cells have been implicated in the pathogenesis of various liver and autoimmune diseases. However, their involvement in primary biliary cholangitis (PBC) has not been elucidated. METHODS: We used immunohistochemistry and flow cytometry to quantify CXCR6+CD8+T cells in hepatic tissue and peripheral blood samples obtained from CXCR6+CD8+T cells obtained from PBC patients. Then, we performed comprehensive statistical analyses to access the correlation between the abundance of these cells and clinical as well as pathological data across different stages of PBC. RESULTS: Our research revealed that CXCR6+ cell frequencies in CD3+CD8+T cells from PBC patients significantly exceeded that of healthy controls (HCs) (2.24 vs. 0.61%, p < 0.01). A similar pattern emerged for hepatic CXCR6+CD8+T cell counts, which were notably higher in the PBC cohort compared to HCs. Our cohort consisted of 118 PBC patients, categorized into 62 early-stage (E-PBC) and 56 late-stage (L-PBC) cases. Notably, significant disparities existed between these groups in terms of liver enzyme and lipid profile levels (p < 0.05), with no notable differences observed in gender, age, blood counts, cholesterol levels, or autoantibodies (p > 0.05). Intriguingly, the quantity of hepatic CXCR6+CD8+T cells per high power field (HPF) was significantly elevated in both E-PBC and L-PBC patients as opposed to normal liver samples, indicating a substantial increase in these cells across all stages of PBC (p = 0.000). Spearman's rank correlation analysis showed a positive correlation between CXCR6+CD8+T cell counts and serum levels of Alkaline Phosphatase (AKP) and Gamma-Glutamyl Transferase (GGT), ANA, IgG and IgM, while revealing a negligible correlation with Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST). Subsequent findings indicated significant variances in CXCR6+ cell numbers not only among different PBC stages but also across various degrees of inflammation and fibrosis (p ≤ 0.007). In a follow-up study post-Ursodeoxycholic Acid (UDCA) treatment, stark differences were identified in biochemical and immunohistochemical profiles between responder (31 patients) and non-responder (33 patients) groups (p < 0.05). A Wilcoxon rank-sum test further demonstrated a significant difference in the level of hepatic CXCR6+CD8+T cells between these two response groups (p = 0.002). CONCLUSION: CXCR6+CD8+T cells play a vital role in the pathogenesis of PBC, exhibiting correlations with the extent of inflammation, staging of liver fibrosis, and response to pharmacological interventions in PBC patients.

Targeting the MHC-I endosomal-lysosomal trafficking pathway in cancer: From mechanism to immunotherapy.

Immune checkpoint blockade (ICB) therapy has achieved broad applicability and durable clinical responses across cancer types. However, the overall response rate remains suboptimal because some patients do not respond or develop drug resistance. The low infiltration of CD8+ cytotoxic T cells (CTLs) in the tumor microenvironment due to insufficient antigen presentation is closely related to the innate resistance to ICB. The duration and spatial distribution of major histocompatibility complex class I (MHC-I) expression on the cell surface is critical for the efficient presentation of endogenous tumor antigens and subsequent recognition and clearance by CTLs. Tumor cells reduce the surface expression of MHC-I via multiple mechanisms to impair antigen presentation pathways and evade immunity and/or develop resistance to ICB therapy. As an increasing number of studies have focused on membrane MHC-I trafficking and degradation in tumor cells, which may impact the effectiveness of tumor immunotherapy. It is necessary to summarize the mechanism regulating membrane MHC-I translocation into the cytoplasm and degradation via the lysosome. We reviewed recent advances in the understanding of endosomal-lysosomal MHC-I transport and highlighted the means exploited by tumor cells to evade detection and clearance by CTLs. We also summarized new therapeutic strategies targeting these pathways to enhance classical ICB treatment and provide new avenues for optimizing cancer immunotherapy.

Nano-Pulse Stimulation Treatment Inhibits Pan02 Murine Pancreatic Tumor Growth and Induces a Long-Term Adaptive Immune Response with Abscopal Effects When Combined with Immune-Enhancing Agents.

Pancreatic cancer is associated with a poor prognosis and immunotherapy alone has not demonstrated sufficient efficacy in the treatment of nonresectable tumors. Nano-Pulse Stimulation™ therapy (NPS™) applies nanosecond electric pulses that lead to regulated cell death, exposing tumor antigen to the immune system. To establish a primary Pan02 tumor, mice were intradermally injected with Pan02 cells into the right flank. Secondary, rechallenge tumors and distal, secondary tumors (abscopal response) were established by injecting Pan02 cells into the opposite left flank. After 5 days of tumor growth, one of the tumors was treated with NPS, followed by injection with an immune-enhancing agent to stimulate an immune response. Growth of the treated primary tumor and untreated rechallenge tumors (injected 60-days post-treatment) or distal secondary tumors (injected simultaneously with the primary) was monitored. NPS in combination with the adjuvant and TLR agonist, resiquimod (RES), was the optimal treatment regimen for both eliminating a primary Pan02 tumor as well as inhibiting growth of a Pan02 cell rechallenge tumor. This inhibition of the rechallenge tumor injected 2 months after eliminating the primary tumor suggests a long-term immune response had been stimulated. Additional support for this came from the observations that depleting CD8+ T-cells reduced inhibition of rechallenge tumor growth by 35% and rechallenge tumors had 3-fold more CD8+ T-cells than tumors injected after surgical resection of the primary tumor. When the NPS-treated tumor was immediately injected with the anti-OX40 antibody to agonize the function of the costimulatory T cell receptor, OX40, up to 80% of untreated abscopal tumors were eliminated. NPS plus RES was the most effective at both eliminating a primary tumor and inhibiting a rechallenge tumor. NPS treatment followed by injection of aOX40 was the most effective at inhibiting the growth of an untreated abscopal tumor.

Long-term use of rituximab increases T cell count in MS patients.

Rituximab has been used to treat MS patients in Iceland for over a decade. However, long-term effect of rituximab on leukocyte populations has not yet been elucidated. By retrospective analysis of flow cytometric data from 349 patients visiting the neurological ward at The National University Hospital of Iceland from 2012 to 2023 for rituximab treatment, the long-term effect of rituximab and whether the effect was dose dependent (1000mg vs 500mg) was evaluated. No difference was detected in efficacy of B cell depletion in patients treated with 500mg as an initial dose of rituximab when compared to 1000mg. Long-term use of rituximab led to an increase in T cell count (p=0,0015) in patients receiving 3-8 doses of rituximab (1.5-8 years of treatment). The increase occurred in both CD4+ (p=0,0028) and CD8+ T cells (p=0,0015) and led to a decrease in the CD4/CD8 ratio (p=0,004). The most notable difference lies in reshaping the balance between näive and effector CD8+ T cells. The clinical implications of long-term treatment with rituximab and its effect on the T cell pool needs to be explored further. Since no difference in B cell depletion was detected between the two patient groups, 1000mg as an initial dose might be excessive, suggesting a personalized dosing regimen might have therapeutic and financial advantages.

Paneth cell differentiation associated with neoadjuvant therapy in esophageal adenocarcinoma.

OBJECTIVES: Paneth cells and Paneth cell metaplasia are well-known in pathology as foundational components in the gastrointestinal system. When within malignant cells (Paneth cell differentiation [PCD]), however, the function and significance of these cells is less well understood. Here, we present findings from the first study focused on PCD in postneoadjuvant esophageal adenocarcinoma (EAC) resection specimens. METHODS: Patients with EAC treated with neoadjuvant chemoradioation and followed by esophagectomy between 2012 and 2018 in our institution were retrospectively evaluated. A tissue microarray was constructed, and special and immunohistochemical stains were performed. RESULTS: A total of 64 cases were collected, of which 8 had PCD, as highlighted by periodic acid-Schiff with diastase staining. Adenocarcinomas with PCD were more commonly seen in patients 60 to 70 years of age and typically had a poorly differentiated morphology, observationally fewer stromal mucinous changes, and less lymph node metastasis. ß-catenin activation induced by neoadjuvant therapy was more frequent in the PCD-positive cases. Patients with PCD-positive disease had low programmed cell death 1 ligand 1 levels, no positive or equivocal ERBB2 (HER2) expression, and low CD8-positive T-cell infiltration; they were also mismatch repair proficient. Patients with PCD-positive disease showed a survival pattern inferior to that of patients with PCD-negative disease. CONCLUSIONS: When induced by neoadjuvant therapy in EAC, PCD is associated with high ß-catenin activation, less expression of targetable biomarkers, and a potentially worse clinical prognosis.

The cytokine Meteorin-like inhibits anti-tumor CD8+ T cell responses by disrupting mitochondrial function.

Tumor-infiltrating lymphocyte (TIL) hypofunction contributes to the progression of advanced cancers and is a frequent target of immunotherapy. Emerging evidence indicates that metabolic insufficiency drives T cell hypofunction during tonic stimulation, but the signals that initiate metabolic reprogramming in this context are largely unknown. Here, we found that Meteorin-like (METRNL), a metabolically active cytokine secreted by immune cells in the tumor microenvironment (TME), induced bioenergetic failure of CD8+ T cells. METRNL was secreted by CD8+ T cells during repeated stimulation and acted via both autocrine and paracrine signaling. Mechanistically, METRNL increased E2F-peroxisome proliferator-activated receptor delta (PPARδ) activity, causing mitochondrial depolarization and decreased oxidative phosphorylation, which triggered a compensatory bioenergetic shift to glycolysis. Metrnl ablation or downregulation improved the metabolic fitness of CD8+ T cells and enhanced tumor control in several tumor models, demonstrating the translational potential of targeting the METRNL-E2F-PPARδ pathway to support bioenergetic fitness of CD8+ TILs.

A nomogram based on circulating CD8+ T cell and platelet-to-lymphocyte ratio to predict overall survival of patients with locally advanced nasopharyngeal carcinoma.

PURPOSE: To explore the influence of circulating lymphocyte subsets, serum markers, clinical factors, and their impact on overall survival (OS) in locally advanced nasopharyngeal carcinoma (LA-NPC). Additionally, to construct a nomogram predicting OS for LA-NPC patients using independent prognostic factors. METHODS: A total of 530 patients with LA-NPC were included in this study. In the training cohort, Cox regression analysis was utilized to identify independent prognostic factors, which were then integrated into the nomogram. The concordance index (C-index) was calculated for both training and validation cohorts. Schoenfeld residual analysis, calibration curves, and decision curve analysis (DCA) were employed to evaluate the nomogram. Kaplan-Meier methods was performed based on risk stratification using the nomogram. RESULTS: A total of 530 LA-NPC patients were included. Multivariate Cox regression analysis revealed that the circulating CD8+T cell, platelet-to-lymphocyte ratio (PLR), lactate dehydrogenase (LDH), albumin (ALB), gender, and clinical stage were independent prognostic factors for LA-NPC (p < 0.05). Schoenfeld residual analysis indicated overall satisfaction of the proportional hazards assumption for the Cox regression model. The C-index of the nomogram was 0.724 (95% CI: 0.669-0.779) for the training cohort and 0.718 (95% CI: 0.636-0.800) for the validation cohort. Calibration curves demonstrated good correlation between the model and actual survival outcomes. DCA confirmed the clinical utility enhancement of the nomogram over the TNM staging system. Significant differences were observed in OS among different risk stratifications. CONCLUSION: Circulating CD8+ T cell, PLR, LDH, ALB, gender and clinical stage are independent prognostic factors for LA-NPC. The nomogram and risk stratification constructed in this study effectively predict OS in LA-NPC.

NKG2D-bispecific enhances NK and CD8+ T cell antitumor immunity.

BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.