Intratumoral CD8 Research Articles

Bivalent OX40 Aptamer and CpG as Dual Agonists for Cancer Immunotherapy.

Cancer immunotherapy has revolutionized cancer treatment by harnessing the body's immune system to recognize and attack tumors. Over the past 25 years, the use of blocking antibodies has fundamentally transformed the landscape of cancer therapy. However, despite extensive research, agonist antibodies targeting costimulatory receptors such as ICOS, GITR, OX40, CD27, and 4-1BB have consistently underperformed in clinical trials over the past 15 years, failing to meet the anticipated success. One reason the agonist antibodies failed is that researchers escalated the dose to the highest tolerable level, which can lead to cell exhaustion, especially when used as a single agent. In this study, we introduced novel in situ therapeutic agents by combining a bivalent RNA aptamer of OX40, biROX40, which binds to two copies of the OX40 receptor as an agonist, with CpG, a toll-like receptor 9 (TLR9) immune stimulator. These agents were specifically designed for lymphoma treatment, with the dose reduced to the lowest bioactive amount to maximize efficacy while minimizing potential side effects. BiROX40 and CpG exhibited a dual immune activation effect and demonstrated a synergistic response even at extremely low dose of 0.32 mg/kg (5.75 μg per mouse) for biROX40 and moderate dose of 1.39 mg/kg (25 μg per mouse) for CpG, resulting in remarkable antitumor efficacy. This effect was achieved through the promotion of intratumoral CD8+ T cell proliferation and cytokine secretion, inhibition of regulatory T cell (Treg) proliferation, and enhanced generation and proliferation of memory T cells in immune organs. The agonistic effects of these reagents led to tumor regression not only at the treated sites but also at distant, nontreated locations in the animal models. This outcome highlighted the induction of a robust systemic antitumor immune response, which effectively suppressed tumor recurrence. This in situ combination therapy, utilizing low-dose biROX40 alongside CpG, offers a straightforward and widely applicable strategy to enhance immune responses in cancer immunotherapy. This approach overcomes the limitations of high-dose single-agent anti-OX40 therapies (whether antibodies or aptamers), including immune cell exhaustion and diminished efficacy.

Tumor-Infiltrating Lymphocytes and Neutrophils and Their Location in Canine Mammary Neoplasms with a Solid Arrangement: A Prognostic Factor?

In canine mammary neoplasms, greater inflammation is associated with higher histological grade, lymphatic invasion, and metastases. This retrospective study assessed the density of peri- and intratumoral tumor-infiltrating lymphocytes (TILs), tumor-associated neutrophils (TANs), and CD3+ and CD79+ lymphocytes in canine mammary neoplasms with a solid arrangement, and associated such data with histological types, immunophenotype, prognostic factors, cyclooxygenase-2 (Cox-2) expression and overall and cancer-specific survival. Sixty-one neoplasms with a solid arrangement were classified as malignant myoepitheliomas (6/9.8%), solid papillary carcinomas (8/13.1%), carcinomas with a solid pattern (9/14.8%), basaloid carcinomas (BC) (19/31.1%), and malignant adenomyoepitheliomas (19/31.1%). Intra- and peritumoral TILs, TANs, and TCD3+ and BCD79+ lymphocytes were counted, and based on the resulting median, the neoplasms were divided into low or high cell infiltration. BCs had the lowest density of intratumoral TILs (p = 0.02), and luminal B neoplasms showed a significantly higher density of intratumoral TCD3+ than luminal A cases. Neoplasms with a higher density of peritumoral CD3+ and CD79+ had significantly greater proliferative activity. High infiltration of intratumoral BCD79+ lymphocytes was related to nodal metastasis (p = 0.03). Intratumoral TILs and TCD3+ were associated with shorter survival time. Therefore, intratumoral lymphocyte infiltration is possibly an important feature in the progression of cancer and influences the survival in bitches with solid arrangement neoplasms.

Comparative evaluation of PD-L1 expression and tumor immune microenvironment in molecular subtypes of muscle-invasive bladder cancer and its correlation with survival outcomes.

Immune checkpoint inhibitors have revolutionized treatment of platinum-refractory advanced bladder cancer, offering hope where options are limited. Response varies, however, influenced by factors such as the tumor's immune microenvironment and prior therapy. Muscle-invasive bladder cancer (MIBC) is stratified into molecular subtypes, with distinct clinicopathologic features affecting prognosis and treatment. This study assessed the expression of programmed cell death 1 ligand 1 (PD-L1) and other immune markers in MIBC, categorized by molecular phenotype. Using GATA3 and CK5/6 immunohistochemistry, 90 neoadjuvant chemotherapy-naive MIBC cases were classified into luminal andnon-luminalsubtypes. The immune microenvironment was characterized through immunostaining for PD-L1, CD4, and CD8. We applied PD-L1 positivity thresholds of 1% or greater for tumor cells and 5% or greater for immune cells. Tumors were examined for PD-L1 expression, histologic subtypes, and immune cell infiltration. Varied expression of PD-L1 and T-cell subtype densities were observed among MIBC subtypes. The double-negative subtype displayed the highest PD-L1 immune cell expression and stromal CD4 and CD8 T-cell densities, indicating an active immune profile. The basal subtype exhibited the highest PD-L1 positivity in tumor cells. In contrast, the luminal type showed the lowest PD-L1 tumor and immune cell expression, with high intratumoral CD4 T-cell density. Although PD-L1 expression in tumor or immune cells did not independently affect survival, patients with basal and double-negative tumors had poorer overall survival. This study highlighted the immune diversity of MIBC in the context of molecular subtypes. Distinct molecular and immune profiles could guide the development of predictive signatures for enhanced immunotherapy response in advanced bladder cancer.

Trabectedin enhances the antitumor effects of IL-12 in triple-negative breast cancer.

Interleukin-12 (IL-12) is a potent NK cell-stimulating cytokine, but the presence of immunosuppressive myeloid cells such as myeloid-derived suppressor cells (MDSC) can inhibit IL 12-induced NK-cell cytotoxicity. Thus, we hypothesized that trabectedin, a myeloid cell-depleting agent, would improve the efficacy of IL-12 in triple-negative breast cancer (TNBC). In vitro treatment of healthy donor NK cells with trabectedin increased expression of the activation marker CD69 and mRNA expression of T BET (Tbx21), the cytotoxic ligands TRAIL (TNFSF10) and Fas ligand (FASLG) and the dendritic cell (DC)-recruiting chemokine lymphotactin (XCL1). The combination of IL-12 and trabectedin increased NK-cell cytotoxicity, activation and production of IFN-γ, TNF-α and granzyme B in the presence of human TNBC cells. Treatment of 4T1 and EMT6 tumor-bearing mice with IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent controls, and the highest levels of plasma IFN-γ, intratumoral CD8+ T cells and conventional type 1 DC. MDSC and M2-like macrophages were significantly decreased with combination therapy. NK-cell depletion abrogated the effects of combination therapy, as did elimination of CD8+ T cells. NK-cell depletion led to lower levels of the NK cell-derived chemokine CCL5 and the DC-derived chemokine CXCL10, higher tumor burden, and decreased intratumoral CD8+ T cells. IL 12 and trabectedin also significantly enhanced the response of TNBC to anti-PD-L1 therapy. These data suggest that MDSC depletion augments the ability of IL-12-activated NK cells to drive the infiltration of DC and CD8+ T cells into TNBC for an antitumor effect.

Highly-Multiplexed Immunofluorescence PhenoCycler Panel for Murine FFPE Yields Insight into Tumor Microenvironment Immunoengineering

Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is Co-Detection-by-indEXing (CODEX), recently renamed as the PhenoCycler system. This is a highly-multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh-frozen tissues. Formalin-fixed paraffin-embedded (FFPE) blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine FFPE tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of three gene-delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift towards intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of IFNγ, in addition to 4-1BBL and IL-12, further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help to explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of IFNγ therapeutic efficacy.

Hepatocellular carcinoma epi-immunotherapy with polyion complex micelles co-delivering HDAC8 inhibitor and PD-L1 siRNA

Hepatocellular carcinoma (HCC) lacking T cell infiltration is a malignancy with a poor prognosis and has an increasing incidence worldwide. Overexpression of histone deacetylase 8 (HDAC8) in HCC caused immune escape and promoted resistance to immune-checkpoint blockade therapy. Herein, we synthesized a polyion complex (PIC) to co-encapsulate the HDAC8 inhibitor PCI-34051 (PCI) and programmed death-ligand 1 small interfering RNA (siPDL1). The PIC micelles PCI-siPDL1@NPs efficiently delivered PCI and siPDL1 into Hepa1-6 cells and orthotopic Hepa1-6 tumors, inducing potent anti-tumor immune responses through a combination of epigenetic therapy and immunotherapy (epi-immunotherapy). PCI-siPDL1@NPs activated T cell-trafficking chemokine C-C motif ligand 4 (CCL4) production via HDAC8 inhibitor and decreased PD-L1 expression via siPDL1. The synergistic epi-immunotherapy significantly increased intratumoral CD8+ T cell infiltration, CD8+ T cell-mediated tumor cell killing, and the immunogenicity of HCC. Furthermore, PCI-siPDL1@NPs produced strong anti-tumor responses in the orthotopic Hepa1-6 HCC model and subcutaneous H22 model, which stimulated tumor regression and prolonged mice survival time. This study successfully constructed stable PIC micelles PCI-siPDL1, which synergistically activated antitumor immunity and reprogrammed the immunosuppressive tumor microenvironment for the treatment of HCC.

IRF1 transcriptionally up-regulates CXCL10 which increases CD8+ T cells infiltration in colorectal cancer.

Tumor-infiltrating CD8+ T cell is a robust predictor of outcome and immunotherapy response in patients with CRC. However, limited introduction of intratumoral CD8+ T cells remains a barrier for treatment of CRC. One of the most effective but difficult therapy for CD8+ T cells entering the tumor is activating chemokine receptors. This study observed a decrease in the expression level of interferon regulator factor 1(IRF1) in CRC tumor tissues compared to matched non-tumor tissues. Furthermore, it found a positive correlation between low IRF1 expression and unfavorable prognosis in CRC patients. The present study also demonstrated that overexpression of IRF1 attenuated tumor growth by promoting the accumulation of facilitating CD8+T cells at the tumor site in mouse models. Additionally, this study identified IRF1 response elements in the promoter region of CXCL10 and show that the binding of IRF1 promoted the transcription of CXCL10. Of note, it was discovered that an increase in CXCL10 was positively associated with improved survival in CRC. These findings strongly suggest that IRF1 serves as a key transcription factor for CXCL10, highlighting its potential as a therapeutic target for CRC.

High levels of fibrotic tumor components are associated with recurrence and intratumoral immune status in advanced colorectal cancer patients

The importance of collagen and elastin remains incompletely understood concerning tumor immunity in cancer tissues. This study explored the clinical significance of collagen and elastin deposition on tumor immunity in advanced colorectal cancer patients. The collagen and elastin contents were assessed simultaneously using elastic van Gieson (EVG) histochemical staining. Immunohistochemical staining was performed to measure the immune cell markers CD3, CD8, CD86, and CD163 in surgically resected primary tumors from 78 pT4 colorectal cancer patients. High collagen, elastin, and EVG scores are associated with aggressive characteristics and short disease-free survival. A high EVG score was identified as an independent predictor of poor disease-free survival. Furthermore, tumors with high collagen and EVG scores exhibited significantly fewer intratumoral CD3 + and CD8 + cells. Evaluating tumor fibrosis using the classical and straightforward EVG staining method could be a reliable predictor of recurrence in high-risk colorectal cancer patients with tumor immune tolerance.

Microbubble-Protected Oncolytic Virotherapy Targeted by Sonoporation Induces Tumor Necrosis and T-Lymphocyte Infiltration in Humanized Mice Bearing Triple-Negative Breast Cancer.

Oncolytic virotherapy has shown great promise in mediating targeted tumor destruction through tumor-selective replication and induction of anti-tumor immunity; however, obstacles remain for virus candidates to reach the clinic. These include avoiding neutralizing antibodies, preventing stimulation of the adaptive immune response during intravenous administration, and inducing sufficient apoptosis and immune activation so that the body's defense can work to eradicate systemic disease. We have developed a co-formulation of oncolytic viruses (OVs) with Imagent® lipid-encapsulated, perfluorocarbon microbubbles (MBs) to protect the OVs from the innate and adaptive immune system. Once inside the MB, the viral particles become acoustically active such that external ultrasound can target the delivery of the virus locally within the tumor. Humanized NSG female mice (Hu-CD34+ NSG-SGM3) engrafted in their flanks with MDA-MB-231-Luc triple-negative breast cancer (TNBC) cells were transduced with MB/OVs, with or without adjuvant Pembrolizumab treatment, and tumor sizes and tumor necrosis were assessed. The presence of CD8+ (cytotoxic T-cells), CD4+ (helper T-cells), and CD25+ (Tregs) tumor-infiltrating lymphocytes (TILs) was quantified in the tumor samples by immunohistochemistry. In an in vivo model of humanized mice engrafted with a human immune system, we observed significantly greater tumor necrosis and smaller tumor mass in human TNBC xenografts systemically treated with MB/OV complexes in the presence or absence of pembrolizumab adjuvant treatment, compared to controls. Additionally, we observed a low ratio of CD4+/CD8+ TILs and a high ratio of CD8+/CD25+ TILs in the MDA-MB-231 xenografts treated with MB/OVs complexes with or without pembrolizumab adjuvant treatment, compared to controls. Our study demonstrated the feasibility of using MBs to target OVs to TNBC through diagnostic ultrasound, which decreased tumor mass by increasing tumor necrosis and stimulated a local and systemic antitumoral immune response by increasing intratumoral CD8+ T-cytotoxic lymphocyte infiltration and decreasing CD25+ Treg cells.

First-Line PD-1 Blockade Combined With Chemotherapy for Stage IV Penile Squamous Cell Carcinoma: A Multicenter Retrospective Study.

The purpose of this study was to evaluate the efficacy and safety of PD-1 blockade combined with cisplatin and paclitaxel (TP)-based chemotherapy as first-line treatment for advanced penile squamous cell carcinoma (PSCC). A retrospective review was performed of 32 eligible patients with high-risk stage IV (cN3M0-1) PSCC who received first-line PD-1 blockade combined with TP-based chemotherapy at 5 medical centers (2019-2023). Clinical responses were assessed using RECIST version 1.1. Treatment-related adverse events (TrAEs) and postsurgical complications were graded according to CTCAE version 5.0. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Multiplex immunofluorescence was used to explore potential biomarkers and to present the tumor microenvironment landscape before and after treatment. After a median treatment duration of 4 cycles (range, 2-6), the overall objective response rate was 78.1% (25/32). Among 27 patients with locally advanced PSCC, 13 (48.1%) subsequently underwent consolidative surgery and 6 (22.2%) achieved a pathologic complete response (pCR). Additionally, 8 (25.0%) patients in the overall cohort underwent consolidated radiotherapy. Median follow-up was 21.1 months (95% CI, 14.1-42.7). Median PFS and OS were 15.0 months (95% CI, 11.4-not available [NA]) and 19.3 months (95% CI, 16.7-NA), respectively. All patients experienced TrAEs, with 50% (16/32) of them having grade ≥3 TrAEs. Higher intratumoral CD8+ T-cell infiltration was observed in pretreatment samples of responders compared with nonresponders (P=.03). CD4+ T-cells, natural killer cells, and macrophages, among others, exhibited significant changes after treatment (all P<.05), suggesting their potential involvement in the antitumor response to immunochemotherapy. PD-1 blockade plus TP-based chemotherapy was effective and well tolerated, with favorable survival outcomes for patients with stage IV PSCC. High pretreatment intratumoral CD8+ T-cell infiltration may help to identify potential responders.

Enabling immune checkpoint blockade efficacy in T-lymphopenia by restoring CD8 T cell dynamics with IL-7 cytokine therapy

IntroductionT-lymphopenia (TLP) is a frequently observed condition in cancer patients, often exacerbated by conventional chemo/radiotherapy, which impairs the efficacy of subsequent immune checkpoint blockade (ICB) therapy. This study aimed to understand the impact of TLP on ICB responsiveness and explore potential therapeutic strategies to enhance antitumor immunity.MethodsTo investigate ICB responsiveness depending on the severity of TLP, first, we established TLP mouse models that mimic clinically observed mild and severe TLP through thymectomy and anti-Thy1-induced peripheral T cell depletion. T cell-replete mice and T-lymphopenic mice were inoculated with palpable or advanced tumors to evaluate the antitumor efficacy of anti-PD-1 therapy according to the severity of TLP. Additionally, by utilizing established murine models, we analyzed matched blood, tumor-draining lymph nodes (TDLNs), and tumor samples by flow cytometry to investigate the mechanisms by which ICB responsiveness is impaired under T-lymphopenic conditions. Finally, to evaluate the combination effect of anti-PD-1 and recombinant IL-7 cytokine therapy (rhIL-7-hyFc) in T-lymphopenic conditions, we administered anti-PD-1, rhIL-7-hyFc, or both to advanced tumor-bearing T-lymphopenic mice and subsequently analyzed tumor growth and survival rates.ResultsUsing mouse models mimicking clinical TLP, we observed that the antitumor efficacy of anti-PD-1 therapy was severely impaired in TLP, depending on the degree of TLP and the immunogenicity of the tumors. TLP mice showed a significant reduction in systemic CD8 T cells but stable intratumoral CD8 T cell numbers, suggesting maintained tumor infiltration despite systemic downregulation. Crucially, TLP led to a shift in the composition of tumor-infiltrating lymphocytes, with a decrease in PD-1+ tumor-reactive CD8 T cells and an increase in PD-1− bystander cells. This reduction in PD-1+ cells was linked to impaired clonal expansion in tumor-draining lymph nodes. To counteract these effects, we introduced recombinant IL-7 cytokine therapy (rhIL-7-hyFc), which effectively restored systemic T cell counts, enhanced PD-1+ CD8 T cell proliferation within tumors, and increased the population of stem-like progenitor cells. The combination of rhIL-7-hyFc and anti-PD-1 therapy resulted in significant tumor regression and improved mouse survival.DiscussionOur findings highlight the critical role of IL-7 in reshaping the CD8 T cell landscape to improve ICB efficacy in TLP conditions, proposing a sequential therapeutic approach: conventional therapy to reduce tumor burden and enhance immunogenicity, followed by IL-7 therapy to restore and rejuvenate CD8 T cells, culminating in effective ICB treatment.

CXCL12-targeted immunomodulatory gene therapy reduces radiation-induced fibrosis in healthy tissues.

Radiation-induced fibrosis (RIF) is a progressive pathology deleteriously impacting cancer survivorship. CXCL12 is an immune-stromal signal implicated in fibrosis and innate response. We hypothesised that modulation of CXCL12 would phenotypically mitigate RIF. CXCL12 expression was characterised in a rodent model of RIF and its expression modulated by the intravascular delivery of lentiviral vectors encoding small hairpin RNA to silence (LVShCXCL12) or overexpress (LVOeCXCL12) CXCL12. Multi-modal fibrotic outcomes were quantified, flow cytometry and Y-chromosome lineage-tracking studies performed to examine cellular recruitment and activation post-radiotherapy (post-RT). Whole-tissue RNA-seq was used to examine matrisomal response. MATBIII tumours were engrafted within tissues with differing levels of CXCL12 expression and tumoral response to RT evaluated. CXCL12 was upregulated in irradiated fibroblasts demonstrating DNA-damage post-RT and led to the recruitment of CD68+ macrophages. Silencing Cxcl12 with LVShCXCL12 demonstrated reduced RIF phenotype as a result of decreased macrophage recruitment. Transcriptomic profiling identified osteopontin (SPP1) as being highly differentially expressed in LVShCXCL12-treated tissues. Tumours growing in tissues devoid of CXCL12 expression responded better following RT due to reductions in peri-tumoural fibrosis as a result of decreased CXCL12 and OPN expression at the tumour/normal tissue interface. This was also associated with greater CD8+ T cell infiltration in tumours with less fibrosis. Antibody-mediated OPN blockade slowed tumour growth by increased intra-tumoral CD8+ T cell activation. The CXCL12/OPN axis is an important node of immune/matrisomal cross-talk in the development of fibrosis. Therapeutic manipulation of this axis may offer greater anti-tumour efficacy whilst also reducing adverse effects.

T cell dynamics with neoadjuvant immunotherapy in head and neck cancer.

Immune-checkpoint inhibitors (ICIs) are being tested as neoadjuvant therapies in various solid tumours, including in patients with head and neck squamous cell carcinoma (HNSCC), with promising results. Key findings thus far include that this approach is well-tolerated with favourable clinical outcomes including promising pathological response rates in initial studies. Pathological responses are likely to be increased by combining other agents with anti-PD-(L)1 antibodies. Comparisons of baseline biopsy samples with post-treatment surgical specimens have enabled correlative studies utilizing multiomic and immunogenomic methods. Data from these studies suggest that pretreatment intratumoural tissue-resident memory CD8+ T cells are key drivers of tumour regression and give rise to both local and systemic antitumour immune responses. Analyses of systemic responses have defined a PD-1+KLRG1- circulating CD8+ T cell subpopulation that is highly predictive of response, and revealed the interrelationships between intratumoural clones and circulating CD8+ T cells. Lastly, interrogation of T cell populations within lymph nodes is beginning to delineate the immune crosstalk between the primary tumour and tumour-draining lymph nodes and how this relationship might be disrupted with tumour infiltration of the latter. In this Review, we examine data from trials testing neoadjuvant ICIs in patients with HNSCC, focusing on human papillomavirus-unrelated disease, and highlight correlative immunogenomic findings from these trials.

CXCR2 expression is associated with prostate-specific membrane antigen expression in hepatocellular carcinoma: reappraisal of tumor microenvironment and angiogenesis.

Angiogenesis is a critical component of neoplastic progression, and inflammatory cells within the tumor microenvironment contribute to neoangiogenesis. Prostate-specific membrane antigen (PSMA)is expressedin the neovasculature of various solid tumors, including hepatocellular carcinoma (HCC). Also, CXCR2 + inflammatory cells, including CD15 + neutrophils, play crucial roles in HCC progression.We evaluated the associations between PSMA expression and CXCR2 + inflammatory cells in HCC by immunohistochemistry (IHC). CXCR2 expression and its correlation with PSMA, the PSMA/CD34 ratio, immune markers (CD3, CD15, CD68, and CD163), clinical parameters, and oncologic outcomes were evaluated in 76HCC and background benign liver tissue. PSMA and the PSMA/CD34 ratioshowed a positive correlationwith intratumoralCXCR2,but not with intratumoral CD15. Intratumoral CXCR2 + cell count was positively associated with intratumoral CD3, CD15, CD68, and CD163 expression levels. In the benign compartment, CXCR2 was significantly associated with CD15. Metabolic dysfunction-associated steatotic liver disease (MASLD) risk factors and cirrhosis had an opposite effect on CXCR2 + cell count in benign liver tissue. Higher CD15 + cell count in the benign liver was associated with decreased overall survival (OS) and recurrence-free survival (RFS). In HCC, intratumoral CXCR2 + cell count is associated with PSMA expression. Intratumoral and benign compartments had different CXCR2 + inflammatory cell makeup. The immune microenvironment of HCC appears to differ depending on underlying risk factors. Further investigations are warranted to elucidate PSMA biology and assess the potential utility of CXCR2 IHC in PSMA-targeted theranostics.

Concurrent intratumoural Treg cell depletion and CD8+ T cell expansion via a cleavable anti-4-1BB-interleukin-15 fusion protein.

Potent agonists of the inducible co-stimulatory receptor 4-1BB are too toxic for patients with advanced cancer. Here, on the basis of observations of a weak agonist of 4-1BB depleting regulatory T (Treg) cells within the tumour microenvironment without leading to substantial restoration of dysfunctional cytotoxic T cells (CTLs), we show that effective tumour control can be achieved via concurrent Treg cell depletion and CTL expansion through an anti-4-1BB antibody fused to interleukin-15 (IL-15) via a peptide sensitive to tumour proteases. In mouse models of advanced cancers, intraperitoneal injection of the bifunctional protein attenuated the activity of the interleukin mostly in the periphery of the primary tumour while allowing for the expansion of CTLs within the tumour microenvironment, led to more effective tumour inhibition and to lower systemic toxicity than treating the cancers with combinatorial treatment with unlinked anti-4-1BB antibody and IL-15, and reduced the resistance of tumours to checkpoint blockade. Concurrent eradication of Treg cells and activation of tumour-infiltrating lymphocytes may represent a general strategy for the effective control of advanced metastatic tumours.

CD3+ Tumor-Infiltrating Lymphocytes in Breast Carcinoma: Their Prognostic Role and Association With Clinical Parameters.

Background Breast carcinoma cases are rising steadily and represent a major causeof mortality and morbidity in India. In response to breast carcinoma, the immune system is activated, resulting in lymphocyteinfiltration in and around the tumor nests. This interaction between the tumor and immune system is the basisfor studyingtumor-infiltrating lymphocytes (TILs). Despite studies on TILs in breast carcinoma, the role of CD3+TILsin predicting patient outcomes remains underexplored. This study aimed to evaluate TILsin breast carcinoma tissues by analyzing CD3 expression through immunohistochemistry (IHC) and examining its association with various prognostic factors, such as histological grade, clinical stage, lymph node status, and hormone receptor status. Methods A study was conducted on 45 breast carcinoma cases, documenting various clinicopathological parameters. IHCstaining was performed for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor (HER2-neu), and CD3 markers on tissue samples. Both intra-tumoral and stromal CD3 TILswere quantified and analyzed in relation to clinicopathological prognostic factors. Results In all 45 breast carcinoma cases, intra-tumoral and stromal CD3 expression was assessed. High stromal CD3 expression is documented in the triple-negative breast tumor. CD3 intra-tumoral expression correlated significantly with tumor size (p < 0.035), ER status (p < 0.036), and PR status (p < 0.036). CD3 stromal expression showed no correlation with any of the prognostic parameters. Conclusion The current study found a strong correlation between CD3 intra-tumoral expression with tumor size, ERand PRstatus. The findings imply that CD3 may be a significant factor in breast carcinoma prognosis. There were more stromal CD3 TILsin triple-negative breast carcinoma (TNBC) cases. TILsought to be examined for their capacity as novel prognostic and predictive indicators, especially in cases of invasive breast cancer, such as triple-negative breast carcinoma.

Lung adenocarcinomas with mucinous histology: clinical, genomic, and immune microenvironment characterization and outcomes to immunotherapy-based treatments and KRASG12C inhibitors

BackgroundApproximately 10% of lung adenocarcinomas (LUAD) have mucinous histology (LUADMuc), which is associated to a light/absent smoking history and a high prevalence of KRAS mutations. We sought to characterize LUADMuc by comparing it to LUAD without mucinous histology (LUADnon-muc) and determine the relative benefit of current treatments. Patients and MethodsPatients with LUAD from five institutions and TCGA PanCancer Atlas classified as LUADMuc or LUADnon-muc were included. Clinicopathologic, genomic, immunophenotypic, transcriptional features, and treatment outcomes were compared between LUADMuc and LUADnon-muc. ResultsOf 4,082 patients with LUAD, 9.9% had LUADMuc. Compared to LUADnon-muc, patients with LUADMuc had a lighter smoking history (median: 15 vs 20 pack-years, P=0.008), lower PD-L1 TPS (median: 0% vs 5%, P<0.0001), and lower tumor mutation burden (median: 6.8 vs 8.5 mutations/megabase, P<0.0001). Mutations in KRAS, NKX2-1 (TTF-1), STK11, SMARCA4, GNAS, and ALK rearrangements were enriched in LUADMuc, while TP53, EGFR, BRAF, and MET mutations were enriched in LUADnon-muc. At stage IV diagnosis, LUADMuc was more likely to have contralateral lung metastasis (55.2% vs 36.9%, P<0.0001) and less likely to have brain metastases (23.3% vs 41.9%, P<0.0001). Compared to LUADnon-muc, LUADMuc cases showed lower intratumor CD8+, PD-1+, CD8+PD-1+, and FOXP3+ cells. Among metastatic cases receiving ICIs, compared to LUADnon-muc (N=1,511), LUADMuc (N=112) had lower objective response rate (ORR, 8.4% vs 25.9%, P<0.0001), and shorter median progression-free survival (mPFS, 2.6 vs 3.9 months, P<0.0001) and overall survival (mOS, 9.9 vs 17.2 months, P<0.0001). Similarly, patients with LUADMuc had worse outcomes to chemo-immunotherapy. LUADMuc (N=18) and LUADnon-muc (N=150) had similar ORR (16.7% vs 34.9%, P=0.12) and mPFS (4.6 vs 5.6 months, P=0.17) to treatment with KRASG12C inhibitors, but LUADMuc had shorter mOS (6.8 vs 10.8 months, P=0.018). ConclusionLUADMuc represents a distinct LUAD subpopulation with unique clinicopathologic, genomic, immunophenotypic, and transcriptional features, achieving worse outcomes to standard immunotherapy-based treatments.

Personalized neoantigen hydrogel vaccine combined with PD-1 and CTLA-4 double blockade elicits antitumor response in liver metastases by activating intratumoral CD8+CD69+ T cells

BackgroundLiver metastasis is highly aggressive and immune tolerant, and lacks effective treatment strategies. This study aimed to develop a neoantigen hydrogel vaccine (NPT-gels) with high clinical feasibility and further investigate...

Flt3L enhances clonal diversification and selective expansion of intratumoral CD8+ T cells while differentiating into effector-like cells

PD-1 blockade enhances anti-tumoral CD8+ Tcell responses via type 1 conventional dendritic cells (cDC1s), but how cDC1s change the properties of intratumoral CD8+ Tcells remains to be determined. Here, we identified two populations of intratumoral CD8+ Tcells distinguished by their expression of asialo-ganglio-N-tetraosylceramide (asGM1). asGM1neg and asGM1posCD8+ Tcells show enriched expression of genes characteristic for precursor exhausted T (Tpex) cells and terminally exhausted T (Tex) cells, respectively. The in situ expression of Flt3L or inhibition of PD-1 each promote the differentiation of asGM1negCD8+ Tcells into asGM1posCD8+ Tcells via interleukin-12 (IL-12) while also increasing the expression of Tpex and effector-like Tcell-associated genes and their effector functions. Both interventions selectively expand CD8+ Tcells, but only Flt3L expression broadens their Tcell receptor (TCR) repertoire. These data indicate the distinct role of Flt3L in diversifying the TCR repertoire, offering potential solutions for immune checkpoint blockade-resistant cancers.

Adjuvant rituximab and elevated intratumoural CD8 expression are associated with sustained disease control after radiotherapy in a randomised trial of systemic therapy in early-stage follicular lymphoma

We report extended follow-up of TROG99.03, a randomised phase III trial in early-stage follicular lymphoma (ESFL) including new information on the role of adjuvant rituximab and translational studies. Patients with ESFL were randomised to involved field radiotherapy (IFRT) or IFRT plus 6-cycles cyclophosphamide/vincristine/prednisolone (IFRT+CVP). From 2006 rituximab was added to IFRT+CVP (IFRT+R-CVP). Clinical and multi-omic parameters were evaluated. Findings were validated in two independent ESFL cohorts (99 and 60 patients respectively). Between 2000 and 2012, 150 (75 per arm) patients were recruited. 48% were positron emission tomography (PET)-staged. By protocol, at median follow-up 11.3-years, progression-free survival (PFS) remained superior for IFRT+(R)CVP vs. IFRT (hazard ratio [HR]=0.60, 95% CI=0.37-0.98, p=0.043; 10-year PFS 62% vs. 43%) respectively. Although no significant difference in overall survival was observed (HR=0.44, 95% CI=0.16-1.18, p=0.11, 10-year OS 95% vs. 84%), patients receiving IFRT+(R)CVP experienced fewer composite (histological transformation and death) events (p=0.045). PFS of IFRT+R-CVP-treated patients compared with all other treatments lacking rituximab (IFRT alone plus IFRT+CVP) was superior (HR=0.36, 95% CI=0.13-0.82, p=0.013). Amongst PET-staged patients, PFS differences between IFRT+R-CVP vs. IFRT were maintained (HR=0.38, 95% CI=0.16-0.89, p=0.027) indicating benefit distinct from stage migration. FL-related mutations and BCL2-translocations were not associated with PFS. However, by multivariate analysis elevated CD8A gene expression in diagnostic biopsy tissue was independently associated with improved PFS (HR=0.45, 95% CI=0.26-0.79, p=0.037), a finding confirmed in both ESFL validation cohorts. CD8A gene expression was raised (p=0.02) and CD8+ T-cell density higher within follicles in ESFL vs. advanced-stage FL (p=0.047). Human leucocyte antigen class I specific neoantigens were detected in 43% of patients, suggesting neoantigen-specific CD8+ T-cells have a role in confining the spread of the disease. Adjuvant R-CVP and elevated intratumoural CD8 expression were independently associated with sustained disease control after radiotherapy in ESFL. Cancer Council Victora; National Health and Medical Research Council; Leukaemia Foundation; Mater Foundation.