Integrative single-cell analysis of longitudinal t(8;21) AML reveals heterogeneous immune cell infiltration and prognostic signatures.

Introduction: Immunotherapies targeting T cells in solid cancers are revolutionizing clinical treatment. Novel immunotherapies have had extremely limited benefit for acute myeloid leukemia (AML). Here, we characterized the immune microenvironment of t(8;21) AML patients to determine how immune cell infiltration status influenced prognosis. Methods: Through multi-omics studies of primary and longitudinal t(8;21) AML samples, we characterized the heterogeneous immune cell infiltration in the tumor microenvironment and their immune checkpoint gene expression. Further external cohorts were also included in this research. Results: CD8+ T cells were enriched and HAVCR2 and TIGIT were upregulated in the CD34+CD117dim%-High group; these features are known to be associated with immune exhaustion. Data integration analysis of single-cell dynamics revealed that a subset of T cells (cluster_2) (highly expressing GZMB, NKG7, PRF1 and GNLY) evolved and expanded markedly in the drug-resistant stage after relapse. External cohort analysis confirmed that the cluster_2 T-cell signature could be utilized to stratify patients by overall survival outcome. Discussion: In conclusion, we discovered a distinct T-cell signature by scRNA-seq that was correlated with disease progression and drug resistance. Our research provides a novel system for classifying patients based on their immune microenvironment.

Tetanus-diphtheria vaccine can prime SARS-CoV-2 cross-reactive T cells.

Vaccines containing tetanus-diphtheria antigens have been postulated to induce cross-reactive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which could protect against coronavirus disease (COVID-19). In this work, we investigated the capacity of Tetanus-diphtheria (Td) vaccine to prime existing T cell immunity to SARS-CoV-2. To that end, we first collected known SARS-CoV-2 specific CD8+ T cell epitopes targeted during the course of SARS-CoV-2 infection in humans and identified as potentially cross-reactive with Td vaccine those sharing similarity with tetanus-diphtheria vaccine antigens, as judged by Levenshtein edit distances (≤ 20% edits per epitope sequence). As a result, we selected 25 potentially cross-reactive SARS-CoV-2 specific CD8+ T cell epitopes with high population coverage that were assembled into a synthetic peptide pool (TDX pool). Using peripheral blood mononuclear cells, we first determined by intracellular IFNγ staining assays existing CD8+ T cell recall responses to the TDX pool and to other peptide pools, including overlapping peptide pools covering SARS-CoV-2 Spike protein and Nucleocapsid phosphoprotein (NP). In the studied subjects, CD8+ T cell recall responses to Spike and TDX peptide pools were dominant and comparable, while recall responses to NP peptide pool were less frequent and weaker. Subsequently, we studied responses to the same peptides using antigen-inexperienced naive T cells primed/stimulated in vitro with Td vaccine. Priming stimulations were carried out by co-culturing naive T cells with autologous irradiated peripheral mononuclear cells in the presence of Td vaccine, IL-2, IL-7 and IL-15. Interestingly, naive CD8+ T cells stimulated/primed with Td vaccine responded strongly and specifically to the TDX pool, not to other SARS-CoV-2 peptide pools. Finally, we show that Td-immunization of C57BL/6J mice elicited T cells cross-reactive with the TDX pool. Collectively, our findings support that tetanus-diphtheria vaccines can prime SARS-CoV-2 cross-reactive T cells and likely contribute to shape the T cell responses to the virus.

cGAS-activated endothelial cell-T cell cross-talk initiates tertiary lymphoid structure formation.

Aberrant activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway causes autoimmunity in humans and mice; however, the exact mechanism by which the cGAS-STING pathway initiates adaptive immunity and tissue pathology is still not fully understood. Here, we used a cGAS knockin (KI) mouse model that develops systemic autoimmunity. In the lungs of cGAS-KI mice, blood vessels were enclosed by organized lymphoid tissues that resemble tertiary lymphoid structures (TLSs). Cell-intrinsic cGAS induction promoted up-regulation of CCR5 in CD8+ T cells and led to CCL5 production in vascular endothelial cells. Peripheral CD8+ T cells were recruited to the lungs and produced CXCL13 and interferon-γ. The latter triggered endothelial cell death, potentiated CCL5 production, and was essential for TLS establishment. Blocking CCL5 or CCR5, or depleting CD8+ T cells, impaired TLS formation. cGAS-mediated TLS formation also enhanced humoral and antitumor responses. These data demonstrate that cGAS signaling drives a specialized lymphoid structure that underlies autoimmune tissue pathology.

Immune Response to COVID-19 Vaccination in Elite Athletes.

Purpose: This study analyses the immune response of elite athletes after COVID-19 vaccination with double-dose mRNA and a single-dose vector vaccine. Methods: Immunoglobulin G (IgG) antibody titers, neutralizing activity, CD4 and CD8 T-cells were examined in blood samples from 72 athletes before and after vaccination against COVID-19 (56 mRNA (BNT162b2 / mRNA-1273), 16 vector (Ad26.COV.2) vaccines). Side effects and training time loss was also recorded. Results: Induction of IgG antibodies (mRNA : 5702 BAU/ml ; 4343 BAU/ml (hereafter: median), vector: 61 BAU/ml ; 52 BAU/ml, p<0.01), their neutralizing activity (99.7% ; 10.6%, p<0.01), and SARS-CoV-2 spike-specific CD4 T-cells (0.13% ; 0.05% ; p<0.01) after mRNA double-dose vaccines was significantly more pronounced than after a single-dose vector vaccine. SARS-CoV-2 spike-specific CD8 T-cell levels after a vector vaccine (0.15%) were significantly higher than after mRNA vaccines (0.02%; p<0.01). When athletes who had initially received the vector vaccine were boostered with an mRNA vaccine, IgG antibodies (to 3456 BAU/ml; p<0.01), neutralizing activity (to 100%; p<0.01), CD4 (to 0.13%; p<0.01) and CD8 T-cells (to 0.43%; p<0.01) significantly increased. When compared with dual-dose mRNA regimen, IgG antibody response was lower (p<0.01), the neutralizing activity (p<0.01) and CD8 T-cell (p<0.01) response higher and no significant difference in CD4 T-cell response (p=0.54) between the two regimens. Cumulative training loss (3 days) did not significantly differ between vaccination regimens (p=0.46). Conclusion: mRNA and vector vaccines against SARSCoV-2 appear to induce different patterns of immune response in athletes. Lower immune induction after a single-shot vector vaccine was clearly optimized by a heterologous booster. Vaccine reactions were mild and short-lived.

Anlotinib potentiates anti-PD1 immunotherapy via transferrin receptor-dependent CD8+ T-cell infiltration in hepatocellular carcinoma.

BACKGROUND: The therapeutic potential of immune checkpoint blockade (ICB) extends across various cancers; however, its effectiveness in treating hepatocellular carcinoma (HCC) is frequently curtailed by both inherent and developed resistance. OBJECTIVE: This research explored the effectiveness of integrating anlotinib (a broad-spectrum tyrosine kinase inhibitor) with programmed death-1 (PD-1) blockade and offers mechanistic insights into more effective strategies for treating HCC. METHODS: Using patient-derived organotypic tissue spheroids and orthotopic HCC mouse models, we assessed the effectiveness of anlotinib combined with PD-1 blockade. The impact on the tumour immune microenvironment and underlying mechanisms were assessed using time-of-flight mass cytometry, RNA sequencing, and proteomics across cell lines, mouse models, and HCC patient samples. RESULTS: The combination of anlotinib with an anti-PD-1 antibody enhanced the immune response against HCC in preclinical models. Anlotinib remarkably suppressed the expression of transferrin receptor (TFRC) via the VEGFR2/AKT/HIF-1α signaling axis. CD8+ T-cell infiltration into the tumour microenvironment correlated with low expression of TFRC. Anlotinib additionally increased the levels of the chemokine CXCL14, crucial for attracting CD8+ T cells. CXCL14 emerged as a downstream effector of TFRC, exhibiting elevated expression following the silencing of TFRC. Importantly, low TFRC expression was also associated with a better prognosis, enhanced sensitivity to combination therapy, and a favourable response to anti-PD-1 therapy in patients with HCC. CONCLUSIONS: Our findings highlight anlotinib's potential to augment the efficacy of anti-PD-1 immunotherapy in HCC by targeting TFRC and enhancing CXCL14-mediated CD8+ T-cell infiltration. This study contributes to developing novel therapeutic strategies for HCC, emphasizing the role of precision medicine in oncology. HIGHLIGHTS: Synergistic effects of anlotinib and anti-PD-1 immunotherapy demonstrated in HCC preclinical models. Anlotinib inhibits TFRC expression via the VEGFR2/AKT/HIF-1α pathway. CXCL14 upregulation via TFRC suppression boosts CD8+ T-cell recruitment. TFRC emerges as a potential biomarker for evaluating prognosis and predicting response to anti-PD-1-based therapies in advanced HCC patients.

Development of an epigenetic clock resistant to changes in immune cell composition.

Epigenetic clocks are age predictors that use machine-learning models trained on DNA CpG methylation values to predict chronological or biological age. Increases in predicted epigenetic age relative to chronological age (epigenetic age acceleration) are connected to aging-associated pathologies, and changes in epigenetic age are linked to canonical aging hallmarks. However, epigenetic clocks rely on training data from bulk tissues whose cellular composition changes with age. Here, we found that human naive CD8+ T cells, which decrease in frequency during aging, exhibit an epigenetic age 15-20 years younger than effector memory CD8+ T cells from the same individual. Importantly, homogenous naive T cells isolated from individuals of different ages show a progressive increase in epigenetic age, indicating that current epigenetic clocks measure two independent variables, aging and immune cell composition. To isolate the age-associated cell intrinsic changes, we created an epigenetic clock, the IntrinClock, that did not change among 10 immune cell types tested. IntrinClock shows a robust predicted epigenetic age increase in a model of replicative senescence in vitro and age reversal during OSKM-mediated reprogramming.

Establishment of a CD8+ T cells-related prognostic risk model for acral melanoma based on single-cell and bulk RNA sequencing.

BACKGROUND: CD8+ T cells have been recognized as crucial factors in the prognosis of melanoma. However, there is currently a lack of gene markers that accurately describe their characteristics and functions in acral melanoma (AM), which hinders the development of personalized medicine. METHODS: Firstly, we explored the composition differences of immune cells in AM using single-cell RNA sequencing (scRNA-seq) data and comprehensively characterized the immune microenvironment of AM in terms of composition, developmental differentiation, function, and cell communication. Subsequently, we constructed and validated a prognostic risk scoring model based on differentially expressed genes (DEGs) of CD8+ T cells using the TCGA-SKCM cohort through Lasso-Cox method. Lastly, immunofluorescence staining was performed to validate the expression of four genes (ISG20, CCL4, LPAR6, DDIT3) in AM and healthy skin tissues as included in the prognostic model. RESULTS: The scRNA-seq data revealed that memory CD8+ T cells accounted for the highest proportion in the immune microenvironment of AM, reaching 70.5%. Cell-cell communication analysis showed extensive communication relationships among effector CD8+ T cells. Subsequently, we constructed a prognostic scoring model based on DEGs derived from CD8+ T cell sources. Four CD8+ T cell-related genes were included in the construction and validation of the prognostic model. Additionally, immunofluorescence results demonstrated that ISG20 and CCL4 were downregulated, while LPAR6 and DDIT3 were upregulated in AM tissues compared to normal skin tissues. CONCLUSION: Identifying biomarkers based on the expression levels of CD8+ T cell-related genes may be an effective approach for establishing prognostic models in AM patients. The independently prognostic risk evaluation model we constructed provides new insights and theoretical support for immunotherapy in AM.

Interaction between microglial cells and CD1C+ B dendritic cells leads to CD8+ T cells depletion during the early stages of renal clear cell carcinoma.

Renal clear cell carcinoma (RCC) is a type of malignant tumor, which, in addition to surgical resection, radiotherapy, and chemotherapy, has been widely treated through immunotherapy recently. However, the influence of the tumor microenvironment and the infiltrating immune cells within it on immunotherapy remains unclear. It is imperative to study the interactions between various immune cells of RCC. The scRNA-seq dataset from GEO's database was used to analyze the immune cells present in tumor tissue and peripheral blood samples. Through quality control, clustering, and identification, the types and proportions of infiltrating immune cells were determined. The cellular differences were determined, and gene expression levels of the differentially present cells were investigated. A protein-protein interaction network analysis was performed using string. KEGG and GO analyses were performed to investigate abnormal activities. The microglia marker CD68 and CD1C+ B dendritic cells marker CD11C were detected using multiplex immunofluorescence staining. Many depleted CD8+ T cells (exhausted CD8+ T cells) appeared in tumor tissues as well as microglia. CD1C+ B dendritic cells did not infiltrate tumor tissues. HSPA1A was correlated with DNAJB1 in microglia. Compared with Paracancer tissues, microglia increased while CD1C+ B dendritic cells decreased in pathological stages I and I-II in cancerous tissues. An altered tumor microenvironment caused by increases in microglia in RCC in the early stage resulted in an inability of CD1C+ B dendritic cells to infiltrate, resulting in CD8+ T cells being unable to receive the antigens presented by them, and in turn being depleted in large quantities.

Rupatadine-inhibited OTUD3 promotes DLBCL progression and immune evasion through deubiquitinating MYL12A and PD-L1.

The obstacle to effectively treating Diffuse Large B-cell Lymphoma (DLBCL) lies in the resistance observed toward standard therapies. Identifying therapeutic targets that prove effective for relapsed or refractory patients poses a significant challenge. OTUD3, a deubiquitinase enzyme, is overexpressed in DLBCL tissues. However, its role in DLBCL has not been investigated. Our study has brought to light the multifaceted impact of OTUD3 in DLBCL. Not only does it enhance cell survival through the deubiquitination of MYL12A, but it also induces CD8+ T cell exhaustion within the local environment by deubiquitinating PD-L1. Our findings indicate that the OTUD3 inhibitor, Rupatadine, exerts its influence through competitive binding with OTUD3. This operation diminishes the deubiquitination of both MYL12A and PD-L1 by OTUD3. This research unveils the central and oncogenic role of OTUD3 in DLBCL and highlights the potential clinical application value of the OTUD3 inhibitor, Rupatadine. These findings contribute valuable insights into addressing the challenges of resistant DLBCL cases and offer a promising avenue for further clinical exploration.

Mutual exclusivity and co-occurrence patterns of immune checkpoints indicate NKG2A relates to anti-PD-1 resistance in gastric cancer.

BACKGROUND: An increasing number of clinical studies have begun to explore combination strategies with immune checkpoint inhibitors, aiming to present new opportunities for overcoming anti-PD-1 treatment resistance in gastric cancer. Unfortunately, the exploration of certain immune checkpoint inhibitor combination strategies has yielded suboptimal results. Therefore, it is necessary to comprehensively analyze the expression patterns of immune checkpoints and identify optimal combination regimens of anti-PD-1 inhibitors with other immune checkpoint inhibitors. METHODS: Leveraging single-cell RNA sequencing (scRNA-seq) and multivariate linear regression interaction models, we dissected the immune checkpoint expression characteristics of CD8+ T cells in gastric cancer and the immune checkpoint expression pattern (ICEP) mediating anti-PD-1 treatment resistance. Furthermore, we employed transcription factor analysis and CellOracle to explore the transcriptional regulatory mechanisms governing CD8+ T cell differentiation fates. Finally, we utilized Nichenet and spatial transcriptomic analysis to investigate the spatial expression patterns of immune checkpoints. RESULTS: Interaction analysis indicated that, among the known immune checkpoints, co-expression of NKG2A and PD-1 might exert a more profound inhibitory effect on the proliferative capacity of CD8+ T cells. The co-expression analysis revealed differential co-expression pattern of PD-1 and NKG2A, defined as ICEP1 (CD8+ T cells co-expressing PD-1, CTLA-4, TIGIT, LAG-3 or CD38) and ICEP2 (CD8+ T cells solely expressing NKG2A or co-expressing with other immune checkpoints), reflecting the co-occurrence pattern of PD-1 and the mutual exclusivity of NKG2A. Further, these two ICEP CD8+ T cell subsets represented distinct CD8+ T cell differentiation fates governed by MSC and RUNX3. Notably, ICEP2 CD8+ T cells were associated with anti-PD-1 therapy resistance in gastric cancer. This phenomenon may be attributed to the recruitment of LGMN+ macrophages mediated by the CXCL16-CXCR6 signaling pathway. CONCLUSION: This study unveiled two distinct ICEPs and the mutually exclusivity and co-occurrence characteristics of CD8+ T cells in gastric cancer. The ICEP2 CD8+ T cell subset, highly expressed in gastric cancer patients resistant to anti-PD-1 therapy, may be recruited by LGMN+ macrophages through CXCL16-CXCR6 axis. These findings provide evidence for NKG2A as a novel immunotherapeutic target in gastric cancer and offer new insights into combination strategies for immune checkpoint inhibitors in gastric cancer.

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.

Tregs delivered post-myocardial infarction adopt an injury-specific phenotype promoting cardiac repair via macrophages in mice.

Regulatory T cells (Tregs) are key immune regulators that have shown promise in enhancing cardiac repair post-MI, although the mechanisms remain elusive. Here, we show that rapidly increasing Treg number in the circulation post-MI via systemic administration of exogenous Tregs improves cardiac function in male mice, by limiting cardiomyocyte death and reducing fibrosis. Mechanistically, exogenous Tregs quickly home to the infarcted heart and adopt an injury-specific transcriptome that mediates repair by modulating monocytes/macrophages. Specially, Tregs lead to a reduction in pro-inflammatory Ly6CHi CCR2+ monocytes/macrophages accompanied by a rapid shift of macrophages towards a pro-repair phenotype. Additionally, exogenous Treg-derived factors, including nidogen-1 and IL-10, along with a decrease in cardiac CD8+ T cell number, mediate the reduction of the pro-inflammatory monocyte/macrophage subset in the heart. Supporting the pivotal role of IL-10, exogenous Tregs knocked out for IL-10 lose their pro-repair capabilities. Together, this study highlights the beneficial use of a Treg-based therapeutic approach for cardiac repair with important mechanistic insights that could facilitate the development of novel immunotherapies for MI.

Influence of cryoablation versus operation on circulating lymphocyte subsets in patients with early-stage renal cell carcinoma.

Immune response is known to play an important role in local tumor control especially in renal cell carcinoma (RCC), which is considered highly immunogenic. For localized tumors, operative resection or local ablative procedures such as cryoablation are common therapeutical options. For thermal ablative procedures such as cryoablation, additional immunological anti-tumor effects have been described.The purpose of this prospective study was to determine changes in peripheral blood circulating lymphocytes and various of their subsets in RCC patients treated with cryoablation or surgery in a longitudinal approach using extensive flow cytometry. Additionally, lymphocytes of RCC patients were compared to a healthy control group.We included 25 patients with RCC. Eight underwent cryoablation and 17 underwent surgery. Univariate and multivariable analysis revealed significantly lower values of B cells, CD4 and CD8 T cells, and various of their subsets in the treatment groups versus the healthy control group. Comparing the two different therapeutical approaches, a significant decline of various lymphocyte subsets with a consecutive normalization after three months was seen for the surgery group, whereas cryoablation led to increased values of CD69 + CD4 + and CD69 + CD8 + cell counts as well as memory CD8 + cells after three months.Treatment-naïve RCC patients showed lower peripheral blood lymphocyte counts compared to healthy controls. The post-treatment course revealed different developments of lymphocytes in the surgery versus cryoablation group, and only cryoablation seems to induce a sustained immunological response after three months.

Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression.

Low response rate, treatment relapse, and resistance remain key challenges for cancer treatment with immune checkpoint blockade (ICB). Here we report that loss of specific tumor suppressors (TS) induces an inflammatory response and promotes an immune suppressive tumor microenvironment. Importantly, low expression of these TSs is associated with a higher expression of immune checkpoint inhibitory mediators. Here we identify, by using in vivo CRISPR/Cas9 based loss-of-function screening, that NF1, TSC1, and TGF-ß RII as TSs regulating immune composition. Loss of each of these three TSs leads to alterations in chromatin accessibility and enhances IL6-JAK3-STAT3/6 inflammatory pathways. This results in an immune suppressive landscape, characterized by increased numbers of LAG3+ CD8 and CD4 T cells. ICB targeting LAG3 and PD-L1 simultaneously inhibits metastatic progression in preclinical triple negative breast cancer (TNBC) mouse models of NF1-, TSC1- or TGF-ß RII- deficient tumors. Our study thus reveals a role of TSs in regulating metastasis via non-cell-autonomous modulation of the immune compartment and provides proof-of-principle for ICB targeting LAG3 for patients with NF1-, TSC1- or TGF-ß RII-inactivated cancers.

Long-term effects on immunological, inflammatory markers, and HIV-1 reservoir after switching to a two-drug versus maintaining a three-drug regimen based on integrase inhibitors.

Objective: To compare the long-term effects on immune parameters, inflammation, and HIV-1 reservoir after switching to a two-drug (2DR) versus maintaining an integrase inhibitor (InSTI)-based three-drug regimen (3DR). Methods: Cross-sectional study in which HIV-1 treatment-naïve people started and maintained an InSTI-based 3DR or, at different times, switched to 2DR (dolutegravir or darunavir/cobicistat + lamivudine). CD4+ and CD8+ T-cell activation and exhaustion, plasma concentrations of hs-CRP, D-dimer, P-selectin, IL-1ß, IL-6, TNF-α, IFN-γ, IP-10, sTNFR-I/II, MIP-1α/ß, I-FABP, LBP, sCD14, sCD163, MCP-1, and cellular-associated HIV-1-DNA and -RNA were quantified by flow cytometry, different immunoassays, and droplet digital PCR, respectively. The U de Mann-Whitney test evaluated differences between 3DR and 2DR. Immune recovery was evaluated using a general linear model for repeated measures adjusted for different co-variables. Results: Fifty participants per group were included. The median time on 3DR was 82 months for the 3DR group and 30 months for the 2DR group, after which it switched to 2DR for a median of 57 months. We did not find differences between both groups in any of the parameters analyzed. Specifically, some values in 3DR and 2DR were hs-CRP, 0.92 mg/L (0.45-2.23) vs. 1.23 (0.61-2.38); D-dimer, 190.0 µg/L (150.0-370.0) vs. 190.0 (150.0-397.5); IL-6, 2.8 pg/mL (1.3-5.3) vs. 3.2 (2.1-4.7); sCD14, 4.5 ng/mL (3.3-6.2) vs. 5.0 (3.6-6.1), respectively, all p ≥ 0.399. Conclusion: In the long term, switching to 2DR does not negatively affect immunologic parameters, inflammatory markers, or HIV-1 reservoir. Clinical trial registration: identifier NCT04076423.

Differential tumor immune microenvironment coupled with tumor progression or tumor eradication in HPV-antigen expressing squamous cell carcinoma (SCC) models.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

Clinical course and prognostic factors of Pneumocystis pneumonia with respiratory failure in non-HIV patients.

Background: Compared with Human Immunodeficiency Virus (HIV) patients, non-HIV patients with Pneumocystis pneumonia (PCP) have more rapid onset, more rapid progression, and higher mortality. Objectives: To investigate the predictive value of variables obtained upon hospital admission for in-hospital death and 90-day outcomes in non-HIV-PCP patients with respiratory failure (RF). Methods: This was a single center retrospective study in a tertiary care institution over 15 years. It included all adults inpatients (≥18 years old) with laboratory confirmed non-HIV-PCP with RF who were discharged or died from Peking University First Hospital between April 1st, 2007 and November 1st, 2022. Epidemiological, clinical, laboratory, imaging and outcome data were collected from patient records. Results: In this study, a total of 146 non-HIV-PCP patients with RF were included. There were 57 patients (39%) died during hospitalization, 44 patients (53%) died in Intensive care unit (ICU). A total of 137 patients completed 90 days of follow-up, of which 58 (42.3%) died. The multivariable regression analysis revealed that a CD8+ T cell count <115/µl (P=0.009), bronchoalveolar lavage fluid (BALF)-neutrophil percentage ≥50% (P=0.047), the time from corticosteroids withdrawal to symptom onset ≤5 days (P=0.012), and the time from visit to initiation of sulfonamides ≥2 days (P=0.011) were independent risk factors for in-hospital death. Furthermore, a CD8+ T cell count < 115/µl (P=0.001) and the time from visit to initiation of sulfonamides therapy ≥2 days (P=0.033) was independently associated with 90-day all-cause death. Conclusions: A low CD8+ T cell count in peripheral blood, a high percentage of BALF-neutrophils, a short time from corticosteroids withdrawal to symptom onset, and a long time from visit to initiation of sulfonamides are associated with poor prognosis in non-HIV-PCP patients with RF.

Impaired Proliferation of CD8+ T Cells Stimulated with Monocyte-Derived Dendritic Cells Previously Matured with Thapsigargin-Stimulated LAD2 Human Mast Cells.

CD8+ T cells are essential for adaptive immunity against infection and tumors. Their ability to proliferate after stimulation is crucial to their functionality. Dendritic cells (DCs) are professional antigen-presenting cells that induce their proliferation. Here, we show that thapsigargin-induced LAD2 mast cell (MC) line-released products can impair the ability of monocyte-derived DCs to induce CD8+ T-cell proliferation and the generation of Th1 cytokine-producing T cells. We found that culture medium conditioned with LAD2 MCs previously stimulated with thapsigargin (thapsLAD2) induces maturation of DCs as determined by the maturation markers CD80, CD83, CD86, and HLA-DR. However, thapsLAD2-matured DCs produced no detectable TNFα or IL-12 during the maturation. In addition, although their surface expression of PD-L1 was comparable with the immature or TLR7/8-agonist (R848)-matured DCs, their TIM-3 expression was significantly higher than in immature DCs and even much higher than in R848-matured DCs. In addition, contrary to R848-matured DCs, the thapsLAD2-matured DCs only tended to induce enhanced proliferation of CD4+ T cells than immature DCs. For CD8+ T cells, this tendency was not even detected because thapsLAD2-matured and immature DCs comparably induced their proliferation, which contrasted with the significantly enhanced proliferation induced by R848-matured DCs. Furthermore, these differences were comparably recapitulated in the ability of the tested DCs to induce IFNγ- and IFNγ/TNFα-producing T cells. These findings show a novel mechanism of MC-mediated regulation of adaptive immune responses.

Peptide-Loaded HLA Class I Tetramer-Associated Magnetic Bead-Based Enrichment of HBV-Specific CD8+ T Cells.

HBV-specific CD8+ T cells are only present at the low frequency during chronic infection. Thus, they are often undetectable by conventional ex vivo staining methods using peptide-loaded HLA class I tetramers. Detection sensitivity can be increased by magnetic bead-based enrichment strategies following staining with peptide-loaded HLA class I tetramers. Additionally, some downstream applications like e.g., single cell RNA sequencing of virus-specific CD8+ T cells may also require a pre-enrichment step to increase the frequency of the cells of interest. For this, peptide-loaded HLA class I tetramers-associated magnetic bead-based enrichment is also a suitable method.

Immunomic longitudinal profiling of the NeoPembrOv trial identifies drivers of immunoresistance in high-grade ovarian carcinoma.

PD-1/PD-L1 blockade has so far shown limited survival benefit for high-grade ovarian carcinomas. By using paired samples from the NeoPembrOv randomized phase II trial (NCT03275506), for which primary outcomes are published, and by combining RNA-seq and multiplexed immunofluorescence staining, we explore the impact of NeoAdjuvant ChemoTherapy (NACT) ± Pembrolizumab (P) on the tumor environment, and identify parameters that correlated with response to immunotherapy as a pre-planned exploratory analysis. Indeed, i) combination therapy results in a significant increase in intraepithelial CD8+PD-1+ T cells, ii) combining endothelial and monocyte gene signatures with the CD8B/FOXP3 expression ratio is predictive of response to NACT + P with an area under the curve of 0.93 (95% CI 0.85-1.00) and iii) high CD8B/FOXP3 and high CD8B/ENTPD1 ratios are significantly associated with positive response to NACT + P, while KDR and VEGFR2 expression are associated with resistance. These results indicate that targeting regulatory T cells and endothelial cells, especially VEGFR2+ endothelial cells, could overcome immune resistance of ovarian cancers.