Characterization of natural killer and cytotoxic T-cell immune infiltrates in pancreatic ductal adenocarcinoma.

BACKGROUND AND OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor response to systemic therapies, including immunotherapy. Given the immunotherapeutic potential of natural killer (NK) cells, we evaluated intratumoral NK cell infiltrates along with cytotoxic T cells in PDAC to determine their association with patient outcomes. METHODS: We analyzed tumors from 93 PDAC patients treated from 2012 to 2020. Predictor variables included tumor-infiltrating lymphocytes (TILs), T-cell markers (CD3, CD8, CD45RO), NK marker (NKp46), and NK inhibitory marker (major histocompatibility complex class I [MHC-I]) by immunohistochemistry. Primary outcome variables were recurrence-free survival (RFS) and overall survival (OS). RESULTS: Mean TILs, CD3, and NKp46 scores were 1.3 ± 0.63, 20.6 ± 17.5, and 3.1 ± 3.9, respectively. Higher expression of CD3 and CD8 was associated with higher OS, whereas NK cell infiltration was not associated with either RFS or OS. There was a tight positive correlation between MHC-I expression and all T-cell markers, but not with NKp46. CONCLUSIONS: Overall NK cell infiltrates were low in PDAC and did not predict clinical outcomes, whereas T-cell infiltrates did. Further characterization of the immune infiltrate in PDAC, including inhibitory signals and suppressive cell types, may yield better biomarkers of prognosis and immune targeting in this refractory disease.

Autoreactive T cells target peripheral nerves in Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a rare heterogenous disorder of the peripheral nervous system, which is usually triggered by a preceding infection, and causes a potentially life-threatening progressive muscle weakness1. Although GBS is considered an autoimmune disease, the mechanisms that underlie its distinct clinical subtypes remain largely unknown. Here, by combining in vitro T cell screening, single-cell RNA sequencing and T cell receptor (TCR) sequencing, we identify autoreactive memory CD4+ cells, that show a cytotoxic T helper 1 (TH1)-like phenotype, and rare CD8+ T cells that target myelin antigens of the peripheral nerves in patients with the demyelinating disease variant. We characterized more than 1,000 autoreactive single T cell clones, which revealed a polyclonal TCR repertoire, short CDR3ß lengths, preferential HLA-DR restrictions and recognition of immunodominant epitopes. We found that autoreactive TCRß clonotypes were expanded in the blood of the same patient at distinct disease stages and, notably, that they were shared in the blood and the cerebrospinal fluid across different patients with GBS, but not in control individuals. Finally, we identified myelin-reactive T cells in the nerve biopsy from one patient, which indicates that these cells contribute directly to disease pathophysiology. Collectively, our data provide clear evidence of autoreactive T cell immunity in a subset of patients with GBS, and open new perspectives in the field of inflammatory peripheral neuropathies, with potential impact for biomedical applications.

Characterization and prognostic impact of ACTBL2-positive tumor-infiltrating leukocytes in epithelial ovarian cancer.

Actin beta-like 2 (ACTBL2) was recently identified as a new mediator of migration in ovarian cancer cells. Yet, its impact on tumor-infiltrating and thus migrating leukocytes (TILs) remains to date unknown. This study characterizes the subset of ACTBL2-expressing TILs in epithelial ovarian cancer (EOC) and elucidates their prognostic influence on the overall survival of EOC patients with special regard to different histological subtypes. Comprehensive immunohistochemical analyses of Tissue-Microarrays of 156 ovarian cancer patients revealed, that a tumor infiltration by ACTBL2-positive leukocytes was significantly associated with an improved overall survival (OS) (61.2 vs. 34.4 months; p = 0.006) and was identified as an independent prognostic factor (HR = 0.556; p = 0.038). This significant survival benefit was particularly evident in patients with low-grade serous carcinoma (OS: median not reached vs. 15.6 months, p < 0.001; HR = 0.058, p = 0.018). In the present cohort, ACTBL2-positive TILs were mainly composed of CD44-positive cytotoxic T-cells (CD8+) and macrophages (CD68+), as depicted by double-immunofluorescence and various immunohistochemical serial staining. Our results provide significant evidence of the prognostic impact and cellular composition of ACTBL2-expressing TILs in EOC. Complementary studies are required to analyze the underlying molecular mechanisms of ACTBL2 as a marker for activated migrating leukocytes and to further characterize its immunological impact on ovarian carcinogenesis.

Copper indium selenium nanomaterials for photo-amplified immunotherapy through simultaneously enhancing cytotoxic T lymphocyte recruitment and M1 polarization of macrophages.

Photoactivated immunotherapy has promising therapeutic efficacy for treating malignancies, especially metastatic tumors. In this study, an erythrocyte membrane-encapsulated copper indium selenium (RCIS) semiconductor nanomaterial was developed to eliminate primary and metastatic tumors, in which copper ions can induce chemodynamic performance, and the narrow band gap endows RCIS with the properties of near-infrared (NIR) light-activated photothermal and photodynamic amplified immunotherapy. Furthermore, RCIS can be used as a nanocarrier to form RNCIS nanoparticles (NPs) by loading NLG919, which blocks the indoleamine 2,3-dioxygenase-1. Under NIR light irradiation, RNCIS NPs release NLG919 at tumor sites via photothermal properties, thereby promoting the recruitment of cytotoxic T lymphocytes and M1 polarization of macrophages, targeting the activation and amplification of immune responses. Herein, in vitro and in vivo studies showed that RNCIS NPs effectively kill cancer cells and eliminate primary and metastatic tumors. Therefore, this study suggests that semiconductor nanomaterials with narrow bandgaps have great potential as photoimmunotherapy agents and NIR light-responsive nanocarriers for controlled release, providing a great paradigm for synergetic tumor photoimmunotherapy. STATEMENT OF SIGNIFICANCE: The Erythrocyte membrane-coated, NLG919-loaded copper indium selenium (RNCIS) semiconductor was designed for eliminating primary and metastatic tumors. RNCIS exhibits chemodynamic, photodynamic, and photothermal activated immunotherapy by inhibiting indoleamine 2,3-dioxygenase-1. This can enhance the recruitment of cytotoxic T lymphocyte and M1 polarization of macrophage, leading to higher synergetic photo-immune therapeutic efficacy.

Targeting TREM1 augments antitumor T cell immunity by inhibiting myeloid-derived suppressor cells and restraining anti-PD-1 resistance.

The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.

Inhibitory Effect of Zinc on Colorectal Cancer by Granzyme B Transcriptional Regulation in Cytotoxic T Cells.

Zinc is one of the essential trace elements and is involved in various functions in the body. Zinc deficiency is known to cause immune abnormalities, but the mechanism is not fully understood. Therefore, we focused our research on tumor immunity to elucidate the effect of zinc on colorectal cancer and its mechanisms. Mice were treated with azoxymethane (AOM) and dextran sodium sulfate (DSS) to develop colorectal cancer, and the relationship between zinc content in the diet and the number and area of tumors in the colon was observed. The number of tumors in the colon was significantly higher in the no-zinc-added group than in the normal zinc intake group, and about half as many in the high-zinc-intake group as in the normal-zinc-intake group. In T-cell-deficient mice, the number of tumors in the high-zinc-intake group was similar to that in the normal-zinc-intake group, suggesting that the inhibitory effect of zinc was dependent on T cells. Furthermore, we found that the amount of granzyme B transcript released by cytotoxic T cells upon antigen stimulation was significantly increased by the addition of zinc. We also showed that granzyme B transcriptional activation by zinc addition was dependent on calcineurin activity. In this study, we have shown that zinc exerts its tumor-suppressive effect by acting on cytotoxic T cells, the center of cellular immunity, and increases the transcription of granzyme B, one of the key molecules in tumor immunity.

Association Between Serum Cytotoxic T Lymphocyte Antigen (CTLA)-4 Level and Disease Progression in Patients With Chronic Hepatitis B.

Backgroud: Immune impairment, marked by increased expression of cytotoxic T lymphocyte antigen (CTLA)-4, promotes the disease progression of chronic hepatitis B. Objective: This study aimed to determine the association between serum CTLA-4 level and disease progression in patients with chronic hepatitis B. Methods: A cross-sectional study was conducted at Haji Adam Malik General Hospital Medan, Indonesia between October 2021 to September 2022. A total of 150 participants were enrolled. Patients aged 18 years or older with evidence of chronic hepatitis B, HBV-related liver cirrhosis, and HBV-related hepatocellular carcinoma (HCC) were enrolled. Exclusion criteria were history of chronic hepatotoxic drug consumption, underlying liver abnormalities other than HBV infection, and liver injury due to metastasized malignancy from other sites. Serum CTLA-4 level was determined from serum using human CTLA-4 enzyme linked immunosorbent assay kit. Results: Most participants were males and aged between 40 and 60 years. Serum CTLA-4 level was positively associated with chronic hepatitis B progression (P<0.001). Serum CTLA-4 level was negatively correlated with serum platelet (P<0.001) and albumin levels (P<0.001) but positively correlated with serum ALT (P=0.045) and total bilirubin levels (P<0.001). Conclusions: Serum CTLA-4 level is associated with disease progression in patients with chronic hepatitis B.

Type 1 interferon signature and cytotoxic T lymphocyte activation targeted against sweat ducts in inflammatory acquired idiopathic generalized anhidrosis.

BACKGROUND: Acquired idiopathic generalized anhidrosis (AIGA) leads to heat intolerance due to the loss or reduction in thermoregulatory sweating over an extensive area of the body. The pathomechanism of AIGA is still unclear but is believed to be autoimmune. OBJECTIVES: We investigated the clinical and pathological features of inflammatory AIGA (InfAIGA) and noninflammatory AIGA (non-InfAIGA) within the skin. METHODS: We compared anhidrotic and normohidrotic skin samples from 30 patients with InfAIGA and non-InfAIGA, as well as skin samples of melanocytic nevus as a negative control. We conducted morphometric analysis and immunohistochemical analysis of cell types and expression of inflammatory molecules (TIA1, CXCR3 and MxA). MxA expression was used as a proxy for type 1 interferon activity. RESULTS: We found that tissue samples from patients with InfAIGA exhibited inflammation within the sweat duct and atrophy of the sweat coil, whereas patients with non-InfAIGA exhibited only atrophy of the sweat coil. Cytotoxic T lymphocyte infiltration and MxA expression were only observed in the sweat ducts of patients with InfAIGA. CONCLUSIONS: InfAIGA is associated with increased sweat duct inflammation and sweat coil atrophy, whereas non-InfAIGA is only associated with sweat coil atrophy. These data suggest that inflammation leads to epithelial destruction of sweat ducts associated with the sweat coil atrophy and subsequent loss of function. Non-InfAIGA may be regarded as a postinflammatory state of InfAIGA. These observations indicate the contribution of both type 1 and type 2 interferons to sweat gland injury. The mechanism involved is similar to the pathomechanism of alopecia areata (AA).

Fusion with type 2 macrophages induces melanoma cell heterogeneity that potentiates immunological escape from cytotoxic T lymphocytes.

Evasion from immunity is a major obstacle to the achievement of successful cancer immunotherapy. Hybrids derived from cell-cell fusion are theoretically associated with tumor heterogeneity and progression by conferring novel properties on tumor cells, including drug resistance and metastatic capacity; however, their impact on immune evasion remains unknown. Here, we investigated the potency of tumor-macrophage hybrids in immune evasion. Hybrids were established by co-culture of a melanoma cell line (A375 cells) and type 2 macrophages. The hybrids showed greater migration ability and greater tumorigenicity than the parental melanoma cells. The hybrids showed heterogeneous sensitivity to New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T-cell receptor-transduced T (TCR-T) cells and two out of four hybrid clones showed less sensitivity to TCR-T compared with the parental cells. An in vitro tumor heterogeneity model revealed that the TCR-T cells preferentially killed the parental cells compared with the hybrids and the survival rate of the hybrids was higher than that of the parental cells, indicating that the hybrids evade killing by TCR-T cells efficiently. Analysis of a single-cell RNA sequencing dataset of patients with melanoma revealed that a few macrophages expressed RNA encoding melanoma differentiation antigens including melan A, tyrosinase, and premelanosome protein, which indicated the presence of hybrids in primary melanoma. In addition, the number of potential hybrids was correlated with a poorer response to immune checkpoint blockade. These results provide evidence that melanoma-macrophage fusion has a role in tumor heterogeneity and immune evasion. © 2023 The Pathological Society of Great Britain and Ireland.

Tumor immune microenvironment (TIME) to enhance antitumor immunity.

The tumor microenvironment is a result of dynamic interaction between different cellular and non-cellular components. In its essence it is not a solo performer, but an ensemble of performers that includes cancer cells, fibroblasts, myo-fibroblasts, endothelial cells and immune cells. The short review highlights important immune infiltrates within the tumor microenvironment that shape cytotoxic t lymphocyte (CTL)-rich immune hot and CTL-deficient immune cold tumors and novel strategies that have potential role in enhancing our immune responses in both immune hot and immune cold tumors.

Response to primary chemoradiotherapy of locally advanced oropharyngeal carcinoma is determined by the degree of cytotoxic T cell infiltration within tumor cell aggregates.

Background: Effective anti-tumor immune responses are mediated by T cells and require organized, spatially coordinated interactions within the tumor microenvironment (TME). Understanding coordinated T-cell-behavior and deciphering mechanisms of radiotherapy resistance mediated by tumor stem cells will advance risk stratification of oropharyngeal cancer (OPSCC) patients treated with primary chemoradiotherapy (RCTx). Methods: To determine the role of CD8 T cells (CTL) and tumor stem cells for response to RCTx, we employed multiplex immunofluorescence stains on pre-treatment biopsy specimens from 86 advanced OPSCC patients and correlated these quantitative data with clinical parameters. Multiplex stains were analyzed at the single-cell level using QuPath and spatial coordination of immune cells within the TME was explored using the R-package Spatstat. Results: Our observations demonstrate that a strong CTL-infiltration into the epithelial tumor compartment (HR for overall survival, OS: 0.35; p<0.001) and the expression of PD-L1 on CTL (HR: 0.36; p<0.001) were both associated with a significantly better response and survival upon RCTx. As expected, p16 expression was a strong predictor of improved OS (HR: 0.38; p=0.002) and correlated with overall CTL infiltration (r: 0.358, p<0.001). By contrast, tumor cell proliferative activity, expression of the tumor stem cell marker CD271 and overall CTL infiltration, regardless of the affected compartment, were not associated with response or survival. Conclusion: In this study, we could demonstrate the clinical relevance of the spatial organization and the phenotype of CD8 T cells within the TME. In particular, we found that the infiltration of CD8 T cells specifically into the tumor cell compartment was an independent predictive marker for response to chemoradiotherapy, which was strongly associated with p16 expression. Meanwhile, tumor cell proliferation and the expression of stem cell markers showed no independent prognostic effect for patients with primary RCTx and thus requires further study.

Neutrophils as key regulators of tumor immunity that restrict immune checkpoint blockade in liver cancer.

OBJECTIVE: Liver cancer is a deadly malignancy associated with high mortality and morbidity. Less than 20% of patients with advanced liver cancer respond to a single anti-PD-1 treatment. The high heterogeneity of neutrophils in the tumor immune microenvironment in liver cancer may contribute to resistance to immune checkpoint blockade (ICB). However, the underlying mechanism remains largely unknown. METHODS: We established an orthotopic liver cancer model by using transposable elements to integrate the oncogenes Myc and KrasG12D into the genome in liver cells from conditional Trp53 null/null mice (pTMK/Trp53-/-). Flow cytometry and immunohistochemistry were used to assess the changes in immune cells in the tumor microenvironment. An ex vivo coculture assay was performed to test the inhibitory effects of tumor-associated neutrophils (TANs) on CD8+ T cells. The roles of neutrophils, T cells, and NK cells were validated through antibody-mediated depletion. The efficacy of the combination of neutrophil depletion and ICB was evaluated. RESULTS: Orthotropic pTMK/Trp53-/- mouse liver tumors displayed a moderate response to anti-Ly6G treatment but not PD-1 blockade. Depletion of neutrophils increased the infiltration of CD8+ T cells and decreased the number of exhausted T cells in the tumor microenvironment. Furthermore, depletion of either CD8+ T or NK cells abrogated the antitumor efficacy of anti-Ly6G treatment. Moreover, the combination of anti-Ly6G with anti-PD-L1 enhanced the infiltration of cytotoxic CD8+ T cells and thereafter resulted in a significantly greater decrease in tumor burden. CONCLUSIONS: Our data suggest that TANs may contribute to the resistance of liver cancer to ICB, and combining TAN depletion with T cell immunotherapy synergistically increases antitumor efficacy.

Microglia-mediated T cell infiltration drives neurodegeneration in tauopathy.

Extracellular deposition of amyloid-ß as neuritic plaques and intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles are two of the characteristic hallmarks of Alzheimer's disease1,2. The regional progression of brain atrophy in Alzheimer's disease highly correlates with tau accumulation but not amyloid deposition3-5, and the mechanisms of tau-mediated neurodegeneration remain elusive. Innate immune responses represent a common pathway for the initiation and progression of some neurodegenerative diseases. So far, little is known about the extent or role of the adaptive immune response and its interaction with the innate immune response in the presence of amyloid-ß or tau pathology6. Here we systematically compared the immunological milieux in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not those with amyloid deposition developed a unique innate and adaptive immune response and that depletion of microglia or T cells blocked tau-mediated neurodegeneration. Numbers of T cells, especially those of cytotoxic T cells, were markedly increased in areas with tau pathology in mice with tauopathy and in the Alzheimer's disease brain. T cell numbers correlated with the extent of neuronal loss, and the cells dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of interferon-γ and PDCD1 signalling both significantly ameliorated brain atrophy. Our results thus reveal a tauopathy- and neurodegeneration-related immune hub involving activated microglia and T cell responses, which could serve as therapeutic targets for preventing neurodegeneration in Alzheimer's disease and primary tauopathies.

Transcriptional and Clonal Characterization of Cytotoxic T Cells in Crescentic Glomerulonephritis.

SIGNIFICANCE STATEMENT: T-cell infiltration is a hallmark of crescentic GN (cGN), often caused by ANCA-associated vasculitis. Pathogenic T-cell subsets, their clonality, and downstream effector mechanisms leading to kidney injury remain to be fully elucidated. Single-cell RNA sequencing and T-cell receptor sequencing revealed activated, clonally expanded cytotoxic CD4 + and CD8 + T cells in kidneys from patients with ANCA-associated cGN. In experimental cGN, kidney-infiltrating CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), which induced apoptosis in renal tissue cells by activation of procaspase-3, and aggravated disease pathology. These findings describe a pathogenic function of (clonally expanded) cytotoxic T cells in cGN and identify GzmB as a mediator and potential therapeutic target in immune-mediated kidney disease. BACKGROUND: Crescentic GN (cGN) is an aggressive form of immune-mediated kidney disease that is an important cause of end stage renal failure. Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis is a common cause. T cells infiltrate the kidney in cGN, but their precise role in autoimmunity is not known. METHODS: Combined single-cell RNA sequencing and single-cell T-cell receptor sequencing were conducted on CD3 + T cells isolated from renal biopsies and blood of patients with ANCA-associated cGN and from kidneys of mice with experimental cGN. Functional and histopathological analyses were performed with Cd8a-/- and GzmB-/- mice. RESULTS: Single-cell analyses identified activated, clonally expanded CD8 + and CD4 + T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Clonally expanded CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), in the mouse model of cGN. Deficiency of CD8 + T cells or GzmB ameliorated the course of cGN. CD8 + T cells promoted macrophage infiltration and GzmB activated procaspase-3 in renal tissue cells, thereby increasing kidney injury. CONCLUSIONS: Clonally expanded cytotoxic T cells have a pathogenic function in immune-mediated kidney disease.

Analysis of Immune Intratumor Heterogeneity Highlights Immunoregulatory and Coinhibitory Lymphocytes as Hallmarks of Recurrence in Stage I Non-Small Cell Lung Cancer.

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non-small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1+ macrophages (CD68+PD-L1+) or antigen-experienced cytotoxic T cells (CD3+CD8+PD-1+) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC.

Nonredundant Upregulation of CD112R (PVRIG) and PD-1 on Cytotoxic T Lymphocytes Located in T Cell Nests of Colorectal Cancer.

Focal T lymphocyte aggregates commonly occur in colorectal cancer; however, their biological significance is unknown. To study focal aggregates of T lymphocytes, a deep learning-based framework for automated identification of T cell accumulations (T cell nests) was developed using CD8, PD-1, CD112R, and Ki67 multiplex fluorescence immunohistochemistry. To evaluate the clinical significance of these parameters, a cohort of 523 colorectal cancers with clinical follow-up data was analyzed. Spatial analysis of locally enriched CD8+ T cell density and cell-to-cell contacts identified T cell nests in the tumor microenvironment of colorectal cancer. CD112R and PD-1 expressions on CD8+ T cells located in T cell nests were found to be elevated compared with those on CD8+ T cells in all other tumor compartments (P < .001 each). Although the highest mean CD112R expression on CD8+ T cells was observed at the invasive margin, the PD-1 expression on CD8+ T cells was elevated in the center of the tumor (P < .001 each). Across all tissue compartments, proliferating CD8+ T cells showed higher relative CD112R and PD-1 expressions than those shown by non-proliferating CD8+ T cells (P < .001 each). Integration of all available spatial and immune checkpoint expression parameters revealed a superior predictive performance for overall survival (area under the curve, 0.65; 95% CI, 0.60-0.70) compared with the commonly used CD8+ tumor-infiltrating lymphocyte density (area under the curve, 0.57; 95% CI, 0.53-0.61; P < .001). Cytotoxic T cells with elevated CD112R and PD-1 expression levels are orchestrated in T cell nests of colorectal cancer and predict favorable patient outcomes, and the spatial nonredundancy underlies fundamental differences between both inhibitory immune checkpoints that provide a rationale for dual anti-CD112R/PD-1 immune checkpoint therapy.

Spontaneous Regression of Swine Melanoma: The Role of Tumour-infiltrating T and NK Cells.

BACKGROUND/AIM: Melanoma is a skin cancer without effective therapy, showing high immunogenicity and mostly partial spontaneous regression (SR). The exact mechanisms of SR are still not well understood; therefore, the use of animal melanoma models is necessary to unravel the immunological processes during SR. MATERIALS AND METHODS: Skin melanoma samples (n=57) and peripheral blood samples (n=57) from the same animals were collected. Melanoma-bearing Libechov Minipigs (MeLiM) aged 3, 4, 6, 8, 10, 12, 20, and 32 weeks were used, and samples were analysed by flow cytometry for detection of immune cell subpopulations. RESULTS: The proportion of CD3-CD8+ (NK) cells in melanoma samples was found to be higher compared to blood samples at 6-8 weeks of age and then at 12 weeks of age. The population of CD4+CD8+ (effector/memory T helper) cells and CD4-CD8+ (cytotoxic T and NK) cells was also increased in melanoma compared to blood samples in 10-32-week-old pigs. The proportion of CD4-CD8+ cells in melanoma samples, then augmented until the 32nd week. On the contrary, the proportion of CD4+CD8- (naive T helper) cells was lower in melanoma samples versus blood samples in 6-32-week-old animals. CONCLUSION: Cytotoxic T cells were the most abundant population of tumour infiltrating immune cells found in MeLiM melanomas of animals aged 10-32 weeks, probably causing the destruction of melanoma cells. Furthermore, the development of specific (adaptive) immune response represented mainly by cytotoxic T cells seems to be crucial for the successful SR of porcine melanoma.

CD161 Characterizes an Inflamed Subset of Cytotoxic T Lymphocytes Associated with Prolonged Survival in Human Papillomavirus-Driven Oropharyngeal Cancer.

Human papillomavirus (HPV)-driven oropharyngeal carcinoma (OPSCC) is distinct from tobacco- or alcohol-associated OPSCC and has a unique immune landscape. Studies have supported the heterogeneity of T cells, accompanied by a broad repertoire of T-cell responses, within tumors driven by HPV infection. However, the phenotype and function of these HPV-related T cells remain unclear. Using a combination of single-cell RNA sequencing, flow cytometry, pharmacologic inhibition, and immunofluorescence staining, we explored the prognostic implication of HPV-related T cells and further validated our findings in two independent cohorts. Cytotoxic T lymphocytes (CTL) within OPSCC displayed a spectrum of transcriptional signatures. Among which, we identified CD161 receptor, encoded by KLRB1, as a potential marker to distinguish the CTL subsets in HPV-positive OPSCC with a divergent evolutionary trajectory. In-depth analysis revealed that CD161+ CTLs exhibited a more robust immune response over the CD161- counterparts and a T cell-inflamed phenotype that could be further reinvigorated by immune-checkpoint blockade. Despite the high expression of exhaustion markers, reinforcement of CD161+ CTL reactivity was expected to boost immune responses, considering their functional reversibility. We further confirmed that the high level of intratumoral CD161+ CTLs associated with a favorable treatment response and prolonged overall survival. Therefore, our research not only provides an insight into the immune landscape of HPV-driven OPSCC but also sheds light on a special subset of CTLs with prognostic and therapeutic significance.

Expression of FOXA1 Is Associated with the Tumor-Infiltrating M2 Macrophage, Cytotoxic T Lymphocyte, and Effect of Chemotherapy in Bladder Cancer.

PURPOSE: Cisplatin-containing combination chemotherapy has been the standard of care since the late 1980s, but the response rate is <50%. Studies have shown that the efficiency of chemotherapy differs among molecular subtypes of bladder cancer. In this study, we aimed to correlate FOXA1, a marker for differentiation of the basal and luminal subtypes, with tumor immune cell infiltration and the effect of chemotherapy in bladder cancer. MATERIALS AND METHODS: Eighty-three patients with bladder cancer treated with chemotherapy were reviewed. Clinicopathological variables for each case were recorded. FOXA1, M2 tumor-associated macrophage (TAM), dendritic cell (DC), and cytotoxic T lymphocyte (CTL) were examined by immunohistochemistry. The relationship between FOXA1, immune cell infiltration, and clinical response to chemotherapy was assessed. RESULTS: The overall objective response rate was 34%. The objective response rate for tumors with lower FOXA1 expression was 58% and for tumors with higher FOXA1 expression was 12%. Tumors with infiltrated M2 TAM proportion <3% had a higher objective response rate compared with infiltrated M2 TAM proportion >3% tumors (46% vs. 21%, p = 0.02). Tumors with infiltrated CTL proportion >5% had a higher objective response rate compared with infiltrated CTL proportion <5% tumors (50% vs. 17%, p = 0.002). DCs showed no significant differences. We found that the objective response rate for tumors with lower FOXA1 expression, proportion <3% M2 TAM infiltration, and proportion >5% CTL infiltration is 82%. Lower FOXA1 expression was associated with low M2 TAM infiltration and high CTL infiltration. CONCLUSIONS: Thus, we showed that in patients with bladder cancer who received chemotherapy, the higher clinical response rate is associated with low FOXA1 expression, low M2 TAM infiltration, and high CTL infiltration.

CD4+ Cytotoxic T Lymphocytes in Cancer Immunity and Immunotherapy.

CD4+ T cells have the ability to differentiate into relatively specialized effector subsets after exposure to innate immune signals. The remarkable plasticity of CD4+ T cells is required to achieve immune responses in different tissues and against various pathogens. Numerous studies have shown that CD4+ T cells can play direct and indispensable roles in protective immunity by killing infected or transformed cells. Although the lineage decision of commitment to the CD4+ or CD8+ cell lineage is once thought to be inflexible, the identification of antigen-experienced CD4+ T cells with cytotoxic activity suggests the existence of unexpected plasticity for these cells. The recognition of CD4+ cytotoxic T lymphocytes (CTLs) and the mechanisms driving the differentiation of this particular cell subset create opportunities to explore the roles of these effector cells in protective immunity and immune-related pathology. CD4+ CTLs are proven to play a protective role in antiviral immunity. Here, the latest investigations on the phenotypic and functional features of CD4+ CTLs and their roles in antitumor immunity and immunotherapy are briefly reviewed.