Protocol for isolation of tumor-derived extracellular vesicles and functional studies on human T cell subsets.

Extracellular vesicles (EVs) enable communication between cells and tissues and are implicated in modulation of tumor immunosuppression. Here, we present a protocol for isolating tumor-derived EVs and assessing their functional influence in cultures with different subsets of human T cells. We describe steps for differential ultracentrifugation, size exclusion chromatography, EVs quantification, and fluorescence-activated cell sorting of human T cells. We then detail procedures for culturing T cells with EVs and using high-resolution spectral flow cytometry phenotyping for the analysis thereof. For complete details on the use and execution of this protocol, please refer to Swatler et al.1 and Swatler et al.2.

The Proportion of Vδ1T Cells in Peripheral Blood Correlated with Prognosis of Sepsis.

BACKGROUND: Sepsis is a serious condition with a high mortality rate, and septic patients often have organ dysfunction, low tissue perfusion and hypoxia, lactic acidosis, oliguria, or functional brain changes. OBJECTIVE: To observe the number and the function of Vδ1T cells in peripheral blood of septic patients, to analyze the clinical significance of detecting Vδ1T cells, and to clarify the correlation of their presence with the prognosis of sepsis. METHODS: The basic data of the septic patients were recorded at admission. The immunosuppressive function-related molecules on the surface of Vδ1T cells were detected, and the immunosuppressive function of Vδ1T cells was also evaluated. RESULTS: Compared with the healthy controls, the proportion of Vδ1T cells in the blood of septic patients significantly decreased (P<0.01). The proportion of Vδ1T cells in septic patients correlated with the patients' condition (P<0.05). The expression of glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) on the surface of Vδ1T cells in the blood of septic patients significantly increased (P<0.01). The increase of Vδ1T cells in septic patients had inhibitory effects on T cell proliferation and interferon (IFN)-γ secretion. These findings implied that the immunosuppression of Vδ1Tcells in the peripheral blood of septic patients was significantly higher than that of the healthy controls (P<0.01). CONCLUSION: Changes in Vδ1T cells in septic patients were closely related to the patient's condition and prognosis.

Intrahepatic CD69+Vδ1 T cells re-circulate in the blood of patients with metastatic colorectal cancer and limit tumor progression.

BACKGROUND: More than 50% of all patients with colorectal cancer (CRC) develop liver metastases (CLM), a clinical condition characterized by poor prognosis and lack of reliable prognostic markers. Vδ1 cells are a subset of tissue-resident gamma delta (γδ) T lymphocytes endowed with a broad array of antitumor functions and showing a natural high tropism for the liver. However, little is known about their impact in the clinical outcomes of CLM. METHODS: We isolated human γδ T cells from peripheral blood (PB) and peritumoral (PT) tissue of 93 patients undergone surgical procedures to remove CLM. The phenotype of freshly purified γδ T cells was assessed by multiparametric flow cytometry, the transcriptional profiles by single cell RNA-sequencing, the functional annotations by Gene Ontology enrichment analyses and the clonotype by γδ T cell receptor (TCR)-sequencing. RESULTS: The microenvironment of CLM is characterized by a heterogeneous immune infiltrate comprising different subsets of γδ tumor-infiltrating lymphocytes (TILs) able to egress the liver and re-circulate in PB. Vδ1 T cells represent the largest population of γδ TILs within the PT compartment of CLM that is greatly enriched in Vδ1 T effector (TEF) cells expressing constitutive high levels of CD69. These Vδ1 CD69+ TILs express a distinct phenotype and transcriptional signature, show high antitumor potential and correlate with better patient clinical outcomes in terms of lower numbers of liver metastatic lesions and longer overall survival (OS). Moreover, intrahepatic CD69+ Vδ1 TILs can egress CLM tissue to re-circulate in PB, where they retain a phenotype, transcriptional signature and TCR clonal repertoires resembling their liver origin. Importantly, even the increased frequencies of the CD69+ terminally differentiated (TEMRA) Vδ1 cells in PB of patients with CLM significantly correlate with longer OS. The positive prognostic score of high frequencies of CD69+ TEMRA Vδ1 cells in PB is independent from the neoadjuvant chemotherapy and immunotherapy regimens administered to patients with CLM prior surgery. CONCLUSIONS: The enrichment of tissue-resident CD69+ Vδ1 TEMRA cells re-circulating at high frequencies in PB of patients with CLM limits tumor progression and represents a new important clinical tool to either predict the natural history of CLM or develop alternative therapeutic protocols of cellular therapies.

Distinct activities of Vδ1+ T-cells upon different cytomegalovirus reactivation status after haematopoietic transplantation.

Cytomegalovirus (CMV) reactivation is the most frequent viral infectious complication correlating to non-relapse mortality after allogeneic haematopoietic cell transplantation (alloHCT). The intrinsic anti-CMV immunity has not been completely elucidated. γδ T-cells have drawn increasing attentions due to their distinct biological features and potential ability against viral infections. Previous studies reported a general association of γδ T-cells or Vδ2-negative γδ T-cells with CMV reactivation. Whereas researches for the direct responses and specific functions of γδ T subsets remain limited, especially in the scenario of alloHCT. Herein, we initially demonstrated that Vδ1+ T-cells directly and independently recognized cell-free CMV and CMV-infected target cells, and inhibited CMV replication in vitro. The anti-CMV effect of Vδ1+ T-cells was partially through TCRγδ, TLR2 and NKG2D receptor pathways. Further investigation about the anti-CMV characteristics of Vδ1+ T-cells was performed in a clinical cohort with different CMV reactivation status after alloHCT. We found that occasional CMV reactivation remarkably increased the recovery levels and stimulated the functional activity of Vδ1+ T-cells. Whereas disability of Vδ1+ T-cells was observed upon refractory CMV reactivation indicating the differential responses of Vδ1+ T-cells under different CMV reactivation status. CXCL10 and IFN-ß that were dramatically induced by occasional CMV reactivation could re-activate the deficient Vδ1+ T-cells from recipients with refractory CMV reactivation. These findings unveiled the distinct activities of Vδ1+ T-cells in anti-CMV immunity after alloHCT and may help develop novel strategies for the treatment of CMV infectious diseases.

COVID-19 Infection in Patients with Comorbidities: Clinical and Immunological Insight.

AIM: Our study's objectives were to study the clinical and laboratory characteristics that may serve as biomarkers for predicting disease severity, IL-10 levels, and frequencies of different T cell subsets in comorbid COVID-19 patients. METHODS: Sixty-two hospitalized COVID-19 patients with comorbidities were assessed clinically and radiologically. Blood samples were collected to assess the T lymphocyte subsets by flow cytometry and IL-10 levels by ELISA. RESULTS: The most common comorbidities observed in COVID-19 patients were diabetes mellitus (DM), hypertension, and malignancies. Common symptoms and signs included fever, cough, dyspnea, fatigue, myalgia, and sore throat. CRP, ferritin, D dimer, LDH, urea, creatinine, and direct bilirubin were significantly increased in patients than controls. Lymphocyte count and CD4+ and CD8+ T-cells were significantly decreased in comorbid COVID-19 patients, and CD25 and CD45RA expression were increased. CD4+ and CD8+ regulatory T cells (Tregs) and IL-10 levels were significantly decreased in patients. CONCLUSIONS: Many parameters were found to be predictive of severity in the comorbid patients in our study. Significant reductions in the levels and activation of CD4+ and CD8+ T-cells were found. In addition, CD4+ and CD8+ Tregs were significant decreased in patients, probably pointing to a prominent role of CD8+ Tregs in dampening CD4+ T-cell activation.

Stratification of chemotherapy-treated stage III colorectal cancer patients using multiplexed imaging and single-cell analysis of T-cell populations.

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Biomarkers for identification of patients at increased risk of disease recurrence following adjuvant chemotherapy are currently lacking. In this study, we assessed immune signatures in the tumor and tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single-cell analysis technology (Cell DIVETM) and evaluated their correlations with patient outcomes. Tissue microarrays (TMAs) with up to three 1 mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin (FOLFOX) chemotherapy. Single sections underwent multiplexed immunofluorescence staining for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and tumor/cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase, and S6). We used annotations and a probabilistic classification algorithm to build statistical models of immune cell types. Images were also qualitatively assessed independently by a Pathologist as 'high', 'moderate' or 'low', for stromal and total immune cell content. Excellent agreement was found between manual assessment and total automated scores (p < 0.0001). Moreover, compared to single markers, a multi-marker classification of regulatory T cells (Tregs: CD3+/CD4+FOXP3+/PD1-) was significantly associated with disease-free survival (DFS) and overall survival (OS) (p = 0.049 and 0.032) of FOLFOX-treated patients. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. These findings were supported by results from an independent FOLFOX-treated cohort of 191 stage III CRC patients, where higher PD1- Tregs were associated with an increase overall survival (p = 0.015) for CD3+/CD4+/FOXP3+/PD1-. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cell types with stronger correlations with outcomes.

Characteristics of Human Peripheral Blood γδ T Cells Expanded With Zoledronate.

BACKGROUND/AIM: This study aimed to investigate the characteristics of human peripheral blood γδ T cells, which were expanded ex vivo in the presence of zoledronate (ZOL). MATERIALS AND METHODS: Human peripheral blood cells were cultured with IL-2 and IL-15 in the presence or absence of ZOL, which was added as a phospho-antigen, and their phenotypes were assessed by flow cytometry. Expanded γδ T cells were transduced with CD19 CAR vector, and the cytotoxicity was evaluated in vitro and in vivo by flow cytometry. RESULTS: Ex vivo expansion did not hamper the expression of activating receptors. Interestingly, ZOL promoted the expression of CD226 (DNAM-1), TRAIL, and FAS-L in the Vδ1 subset of γδ T cells. Expanded γδ T cells containing CD19 CAR+ γδ T cells removed B cell lymphoma cells effectively in vivo. CONCLUSION: γδ T cells could be a promising immunotherapeutic for cancer.

Circulating Immunological Biomarkers: Prognosis of Pancreatic Cancer Patients Reflected by the Immune System.

ABSTRACT: To date, little advances have been made toward new and more effective therapies for pancreatic ductal adenocarcinoma (PDAC). Discovery of prognostic and predictive biomarkers is needed to stratify patients for available treatments and to elucidate how new therapies could be developed. Recent studies have made clear that the immune system is not only affected in the microenvironment of the primary tumor and it is also systemically disrupted in PDAC patients. Under normal circumstances, the immune system is in perfect balance with both proinflammatory and anti-inflammatory components present. In this review, we focus on circulating immunological characteristics including immune cells and their subtypes, cytokines, and immune checkpoints in the peripheral blood not only to understand the poor prognosis of PDAC patients but also to find new leads for new innovative therapies.

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients.

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer.

Multiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of published studies are limited to the analysis of human patient samples. Performing mIHC on formalin-fixed paraffin-embedded (FFPE) mouse tissues, particularly with sensitive antigens, remain challenging. The aim of our study was to develop a robust and reproducible protocol to uncover the immune landscape in mouse FFPE tissues. Effective antibody stripping while maintaining sensitivity to antigens and tissue adhesion to the glass slide is critical in developing an mIHC panel to allow successive rounds of staining. Thus, we identified a highly efficient stripping method that preserves signal intensity and antigenicity to allow multiple rounds of staining. We subsequently optimised an mIHC workflow with antibodies specific against CD4, CD8α, FOXP3 and B220 to identify distinct T and B cell populations on mouse FFPE tissues. Lastly, the application of this mIHC panel was validated in a mouse model of inflammatory bowel cancer, two allograft mouse models of spontaneous colon adenocarcinoma and a sporadic mouse model of colon cancer. Together, these demonstrate the utility of the aforementioned protocol in establishing the quantity and spatial localisation of immune cells in different pathological tissues.

The Potential of Tissue-Resident Memory T Cells for Adoptive Immunotherapy against Cancer.

Tissue-resident memory T cells (TRM) comprise an important memory T cell subset that mediates local protection upon pathogen re-encounter. TRM populations preferentially localize at entry sites of pathogens, including epithelia of the skin, lungs and intestine, but have also been observed in secondary lymphoid tissue, brain, liver and kidney. More recently, memory T cells characterized as TRM have also been identified in tumors, including but not limited to melanoma, lung carcinoma, cervical carcinoma, gastric carcinoma and ovarian carcinoma. The presence of these memory T cells has been strongly associated with favorable clinical outcomes, which has generated an interest in targeting TRM cells to improve immunotherapy of cancer patients. Nevertheless, intratumoral TRM have also been found to express checkpoint inhibitory receptors, such as PD-1 and LAG-3. Triggering of such inhibitory receptors could induce dysfunction, often referred to as exhaustion, which may limit the effectiveness of TRM in countering tumor growth. A better understanding of the differentiation and function of TRM in tumor settings is crucial to deploy these memory T cells in future treatment options of cancer patients. The purpose of this review is to provide the current status of an important cancer immunotherapy known as TIL therapy, insight into the role of TRM in the context of antitumor immunity, and the challenges and opportunities to exploit these cells for TIL therapy to ultimately improve cancer treatment.

Stem cell-like memory T cells: The generation and application.

Stem cell-like memory T cells (Tscm), are a newly defined memory T cell subset with characteristics of long life span, consistent self-renewing, rapid differentiation into effector T cells, and apoptosis resistance. These features indicate that Tscm have great therapeutic or preventive purposes, including being applied in chimeric Ag receptor-engineered T cells, TCR gene-modified T cells, and vaccines. However, the little knowledge about Tscm development restrains their applications. Strength and duration of TCR signaling, cytokines and metabolism in the T cells during activation all influence the Tscm development via regulating transcriptional factors and cell signaling pathways. Here, we summarize the molecular and cellular pathways involving Tscm differentiation, and its clinical application for cancer immunotherapy and prevention.

Changes in T Lymphocyte Subsets in Different Tumors Before and After Radiotherapy: A Meta-analysis.

Purpose: Radiation therapy (RT) induces an immune response, but the relationship of this response with tumor type is not fully understood. This meta-analysis further elucidated this relationship by analyzing the changes in T lymphocyte subsets in different tumors before and after radiotherapy. Methods: We searched English-language electronic databases including PubMed, EMBASE, and the Cochrane Library to collect studies on the changes in peripheral blood CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes before and after radiotherapy in tumor patients from January 2015 to April 2021. The quality of the included literature was evaluated using the NOS scale provided by the Cochrane Collaboration, and statistical software RevMan 5.4 was used to analyze the included literature. P<0.05 was considered to indicate statistical significance. Results: A total of 19 studies in 16 articles involving 877 tumor patients were included. All data were collected within 1 month before or after radiotherapy. Meta-analysis showed that numbers of CD3+ T lymphocytes (SMD: -0.40; 95% CI [-0.75, -0.04]; p = 0.03) and CD4+ T lymphocytes (SMD: -0.43; 95% CI: [-0.85, -0.02]; p = 0.04) were significantly reduced after radiotherapy compared with before treatment, but there was no statistically significant difference for CD8+ T lymphocytes (SMD: 0.33; 95% CI: [-0.88, 0.74]; p = 0.12). Subgroup analysis showed that peripheral blood T lymphocytes decreased in head and neck cancer. However, in prostate cancer and breast cancer, there was no significant change in peripheral blood. 1 month after radiotherapy, it has a potential proliferation and activation effect on lymphocytes in esophageal cancer and lung cancer. The results showed that CD8+T lymphocytes increased in peripheral blood after SBRT. Radiotherapy alone reduced CD3+ T lymphocyte numbers. Conclusions: Within 1 month of radiotherapy, patients have obvious immunological changes, which can cause apoptosis and reduction of T lymphocytes, and affect the balance of peripheral blood immune cells. The degree of immune response induced by radiotherapy differed between tumor types.

Role of IL-6 in the commitment of T cell subsets.

IL-6 gained much attention with the discovery that this cytokine is a non-redundant differentiation factor for Th17 cells and T follicular helper cells. Adaptive immune responses to fungi and extracellular bacteria are impaired in the absence of IL-6. IL-6 is also required for the induction of ROR-γt+ Treg cells, which are gatekeepers of homeostasis in the gut lamina propria in the presence of commensal bacteria. Conversely, severe immunopathology in T cell-mediated autoimmunity is mediated by Th17 cells that rely on IL-6 for their generation and maintenance. Recently, it has been discovered that the differentiation of these distinct T helper cell subsets may be linked to distinct signaling modalities of IL-6. Here, we summarize the current knowledge on the mode of action of IL-6 in the differentiation and maintenance of T cell subsets and propose that a context-dependent understanding of the impact of IL-6 on T cell subsets might inform rational IL-6-directed interventions in autoimmunity and chronic inflammation.

Mitochondrial transcription factor A in RORγt+ lymphocytes regulate small intestine homeostasis and metabolism.

RORγt+ lymphocytes, including interleukin 17 (IL-17)-producing gamma delta T (γδT17) cells, T helper 17 (Th17) cells, and group 3 innate lymphoid cells (ILC3s), are important immune regulators. Compared to Th17 cells and ILC3s, γδT17 cell metabolism and its role in tissue homeostasis remains poorly understood. Here, we report that the tissue milieu shapes splenic and intestinal γδT17 cell gene signatures. Conditional deletion of mitochondrial transcription factor A (Tfam) in RORγt+ lymphocytes significantly affects systemic γδT17 cell maintenance and reduces ILC3s without affecting Th17 cells in the gut. In vivo deletion of Tfam in RORγt+ lymphocytes, especially in γδT17 cells, results in small intestine tissue remodeling and increases small intestine length by enhancing the type 2 immune responses in mice. Moreover, these mice show dysregulation of the small intestine transcriptome and metabolism with less body weight but enhanced anti-helminth immunity. IL-22, a cytokine produced by RORγt+ lymphocytes inhibits IL-13-induced tuft cell differentiation in vitro, and suppresses the tuft cell-type 2 immune circuit and small intestine lengthening in vivo, highlighting its key role in gut tissue remodeling.

BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection.

During chronic viral infection, CD8+ T cells develop into three major phenotypically and functionally distinct subsets: Ly108+TCF-1+ progenitors, Ly108-CX3CR1- terminally exhausted cells and the recently identified CX3CR1+ cytotoxic effector cells. Nevertheless, how CX3CR1+ effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX3CR1+ effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX3CR1+ subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1+ progenitors to CX3CR1+ effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer.

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

ILC-You in the Thymus: A Fresh Look at Innate Lymphoid Cell Development.

The discovery of innate lymphoid cells (ILCs) has revolutionized our understanding of innate immunity and immune cell interactions at epithelial barrier sites. Their presence and maintenance are critical for modulating immune homeostasis, responding to injury or infection, and repairing damaged tissues. To date, ILCs have been defined by a set of transcription factors, surface antigens and cytokines, and their functions resemble those of three major classes of helper T cell subsets, Th1, Th2 and Th17. Despite this, the lack of antigen-specific surface receptors and the notion that ILCs can develop in the absence of the thymic niche have clearly set them apart from the T-cell lineage and promulgated a dogma that ILCs develop directly from progenitors in the bone marrow. Interestingly however, emerging studies have challenged the BM-centric view of adult ILC development and suggest that ILCs could arise neonatally from developing T cell progenitors. In this review, we discuss ILC development in parallel to T-cell development and summarize key findings that support a T-cell-centric view of ILC ontogeny.

Aberrant cytokine expression in COVID-19 patients: Associations between cytokines and disease severity.

Cytokines play pleiotropic, antagonistic, and collaborative in viral disease. The high morbidity and mortality of coronavirus disease 2019 (COVID-19) make it a significant threat to global public health. Elucidating its pathogenesis is essential to finding effective therapy. A retrospective study was conducted on 71 patients hospitalized with COVID-19. Data on cytokines, T lymphocytes, and other clinical and laboratory characteristics were collected from patients with variable disease severity. The effects of cytokines on the overall survival (OS) and event-free survival (EFS) of patients were analyzed. The critically severe and severe patients had higher infection indexes and significant multiple organ function abnormalities than the mild patients (P < 0.05). IL-6 and IL-10 were significantly higher in the critically severe patients than in the severe and mild patients (P < 0.05). IL-6 and IL-10 were closely associated with white blood cells, neutrophils, T lymphocyte subsets, D-D dimer, blood urea nitrogen, complement C1q, procalcitonin C-reactive protein. Moreover, the IL-6 and IL-10 levels were closely correlated to dyspnea and dizziness (P < 0.05). The patients with higher IL-10 levels had shorter OS than the group with lower levels (P < 0.05). The older patients with higher levels of single IL-6 or IL-10 tended to have shorter EFS (P < 0.05), while the patients who had more elevated IL-6 and IL-10 had shorter OS (P < 0.05). The Cox proportional hazard model revealed that IL-6 was the independent factor affecting EFS. IL-6 and IL-10 play crucial roles in COVID-19 prognosis.

Single-cell analysis reveals the origins and intrahepatic development of liver-resident IFN-γ-producing γδ T cells.

γδ T cells are heterogeneous lymphocytes located in various tissues. However, a systematic and comprehensive understanding of the origins of γδ T cell heterogeneity and the extrathymic developmental pathway associated with liver γδ T cells remain largely unsolved. In this study, we performed single-cell RNA sequencing (scRNA-seq) to comprehensively catalog the heterogeneity of γδ T cells derived from murine liver and thymus samples. We revealed the developmental trajectory of γδ T cells and found that the liver contains γδ T cell precursors (pre-γδ T cells). The developmental potential of hepatic γδ T precursor cells was confirmed through in vitro coculture experiments and in vivo adoptive transfer experiments. The adoptive transfer of hematopoietic progenitor Lin-Sca-1+Mac-1+ (LSM) cells from fetal or adult liver samples to sublethally irradiated recipients resulted in the differentiation of liver LSM cells into pre-γδ T cells and interferon-gamma+ (IFN-γ+) but not interleukin-17a+ (IL-17a+) γδ T cells in the liver. Importantly, thymectomized mouse models showed that IFN-γ-producing γδ T cells could originate from liver LSM cells in a thymus-independent manner. These results suggested that liver hematopoietic progenitor LSM cells were able to differentiate into pre-γδ T cells and functionally mature γδ T cells, which implied that these cells are involved in a distinct developmental pathway independent of thymus-derived γδ T cells.