Porcine γδ T cells express cytotoxic cell-associated markers and display killing activity but are not selectively cytotoxic against PRRSV- or swIAV-infected macrophages.

Background: Gamma-delta (γδ) T cells are a major immune cell subset in pigs. Approximately 50% of circulating T cells are γδ T cells in young pigs and up to 30% in adult sows. Despite this abundance, the functions of porcine γδ T cells are mostly unidentified. In humans and mice, activated γδ T cells exhibit broad innate cytotoxic activity against a wide variety of stressed, infected, and cancerous cells through death receptor/ligand-dependent and perforin/granzyme-dependent pathways. However, so far, it is unknown whether porcine γδ T cells have the ability to perform cytotoxic functions. Methods: In this study, we conducted a comprehensive phenotypic characterization of porcine γδ T cells isolated from blood, lung, and nasal mucosa. To further analyze the cytolytic potential of γδ T cells, in vitro cytotoxicity assays were performed using purified γδ T cells as effector cells and virus-exposed or mock-treated primary porcine alveolar macrophages as target cells. Results: Our results show that only CD2+ γδ T cells express cytotoxic markers (CD16, NKp46, perforin) with higher perforin and NKp46 expression in γδ T cells isolated from lung and nasal mucosa. Moreover, we found that γδ T cells can exhibit cytotoxic functions in a cell-cell contact and degranulation-dependent manner. However, porcine γδ T cells did not seem to specifically target Porcine Reproductive and Respiratory Syndrome Virus or swine Influenza A Virus-infected macrophages, which may be due to viral escape mechanisms. Conclusion: Porcine γδ T cells express cytotoxic markers and can exhibit cytotoxic activity in vitro. The specific mechanisms by which porcine γδ T cells recognize target cells are not fully understood but may involve the detection of cellular stress signals.

Prdm1 positively regulates liver Group 1 ILCs cancer immune surveillance and preserves functional heterogeneity.

Group 1 innate lymphoid cells (ILCs) comprise conventional natural killer (cNK) cells and type 1 innate lymphoid cells (ILC1s). The main functions of liver cNK cells and ILC1s not only include directly killing target cells but also regulating local immune microenvironment of the liver through the secretion of cytokines. Uncovering the intricate mechanisms by which transcriptional factors regulate and influence the functions of liver cNK cells and ILC1s, particularly within the context of liver tumors, presents a significant opportunity to amplify the effectiveness of immunotherapies against liver malignancies. Using Ncr1-drived conditional knockout mouse model, our study reveals the regulatory role of Prdm1 in shaping the composition and maturation of cNK cells. Although Prdm1 did not affect the killing function of cNK cells in an in vivo cytotoxicity model, a significant increase in cancer metastasis was observed in Prdm1 knockout mice. Interferon-gamma (IFN-γ), granzyme B, and perforin secretion decreased significantly in Prdm1-deficient cNK cells and liver ILC1s. Single-cell RNA sequencing (scRNA-seq) data also provided evidences that Prdm1 maintains functional subsets of cNK cells and liver ILC1s and facilitates communications between cNK cells, liver ILC1s, and macrophages. The present study unveiled a novel regulatory mechanism of Prdm1 in cNK cells and liver ILC1s, showing promising potential for developing innovative immune therapy strategies against liver cancer.

CD8+ T Cell Biology in Cytokine Storm Syndromes.

Familial forms of hemophagocytic lymphohistiocytosis (HLH) are caused by loss-of-function mutations in genes encoding perforin as well as those required for release of perforin-containing cytotoxic granule constituent. Perforin is expressed by subsets of CD8+ T cells and NK cells, representing lymphocytes that share mechanism of target cell killing yet display distinct modes of target cell recognition. Here, we highlight recent findings concerning the genetics of familial HLH that implicate CD8+ T cells in the pathogenesis of HLH and discuss mechanistic insights from animal models as well as patients that reveal how CD8+ T cells may contribute to or drive disease, at least in part through release of IFN-γ. Intriguingly, CD8+ T cells and NK cells may act differentially in severe hyperinflammatory diseases such as HLH. We also discuss how CD8+ T cells may promote or drive pathology in other cytokine release syndromes (CSS). Moreover, we review the molecular mechanisms underpinning CD8+ T cell-mediated lymphocyte cytotoxicity, key to the development of familial HLH. Together, recent insights to the pathophysiology of CSS in general and HLH in particular are providing promising new therapeutic targets.

Overexpression of LAG-3: a potential indicator of low immune function in tuberculosis.

Background: Tuberculosis (TB) persists as a global health challenge, with its treatment hampered by the side effects of long-term combination drug therapies and the growing issue of drug resistance. Therefore, the development of novel therapeutic strategies is critical. This study focuses on the role of immune checkpoint molecules (ICs) and functions of CD8+ T cells in the search for new potential targets against TB. Methods: We conducted differential expression genes analysis and CD8+ T cell functional gene analysis on 92 TB samples and 61 healthy individual (HI) samples from TB database GSE83456, which contains data on 34,603 genes. The GSE54992 dataset was used to validated the findings. Additionally, a cluster analysis on single-cell data from primates infected with mycobacterium tuberculosis and those vaccinated with BCG was performed. Results: The overexpression of LAG-3 gene was found as a potentially important characteristic of both pulmonary TB (PTB) and extrapulmonary TB (EPTB). Further correlation analysis showed that LAG-3 gene was correlated with GZMB, perforin, IL-2 and IL-12. A significant temporal and spatial variation in LAG-3 expression was observed in T cells and macrophages during TB infection and after BCG vaccination. Conclusion: LAG-3 was overexpressed in TB samples. Targeting LAG-3 may represent a potential therapeutic target for tuberculosis.

Evidence of membranolytic targeting and intracellular citrullination in neutrophils isolated from patients with rheumatoid arthritis.

Anti-citrullinated protein autoantibodies (ACPA) are diagnostic for rheumatoid arthritis (RA). The antigens recognized by these autoantibodies are produced by protein arginine deiminases (PADs), particularly PAD4. However, it remains unknown why and how PAD4 causes this aberrant citrullination in RA. Here, we report that poly-perforin pores are present on freshly isolated neutrophils from RA patients, but not on healthy donor neutrophils. Neutrophils with perforin pores also contained intracellular citrullinated proteins in the region adjacent to the pores. This response was replicated in vitro by treating neutrophils with purified perforin, which generated intense dots of anti-perforin immunofluorescence, calcium influx, and intracellular citrullination. Extensive neutrophil killing in Felty's syndrome, an aggressive form of RA, correlated with particularly high ACPA, and PAD4 autoantibodies. In contrast, other forms of death, including NETosis, apoptosis, and pyroptosis, produced minimal citrullination. We conclude that neutrophil targeting by perforin leading to intracellular citrullination takes place in patients with RA.

ICOS-expressing Regulatory T Cells Influence the Composition of Antitumor CTL Populations.

The role of ICOS in antitumor T cell responses and overall tumor progression has been controversial. In this study, we compared tumor progression in mice lacking ICOS selectively in regulatory T (Treg) cells or in all T cells. Using an experimental melanoma lung metastasis model, we found that Treg cell-specific ICOS knockout reduces the overall tumor burden compared with Cre control mice, with increased CD4+-to-Treg cell and CD8+-to-Treg cell ratios in the tumor. In contrast, there was no difference in the tumor burden in mice lacking ICOS in all of the T cell compartments. This suggests a dual role of ICOS costimulation in promoting protumor and antitumor T cell responses. Consistent with reduced tumor burden, we found that Treg cell-specific deletion of ICOS leads to an increase of CD8+ CTLs that express high levels of granzyme B and perforin. Moreover, single-cell transcriptome analysis revealed an increase of Ly108+Eomeshi CD8+ T cells at the cost of the Ly108+T-bethi subset in Treg cell-specific knockout mice. These results suggest that ICOS-expressing Treg cells suppress the CTL maturation process at the level of Eomes upregulation, a critical step known to drive perforin expression and cytotoxicity. Collectively, our data imply that cancer immunotherapies using ICOS agonist Abs may work better in Treg cell-low tumors or when they are combined with regimens that deplete tumor-infiltrating Treg cells.

An enterococcal phage-derived enzyme suppresses graft-versus-host disease.

Changes in the gut microbiome have pivotal roles in the pathogenesis of acute graft-versus-host disease (aGVHD) after allogenic haematopoietic cell transplantation (allo-HCT)1-6. However, effective methods for safely resolving gut dysbiosis have not yet been established. An expansion of the pathogen Enterococcus faecalis in the intestine, associated with dysbiosis, has been shown to be a risk factor for aGVHD7-10. Here we analyse the intestinal microbiome of patients with allo-HCT, and find that E. faecalis escapes elimination and proliferates in the intestine by forming biofilms, rather than by acquiring drug-resistance genes. We isolated cytolysin-positive highly pathogenic E. faecalis from faecal samples and identified an anti-E. faecalis enzyme derived from E. faecalis-specific bacteriophages by analysing bacterial whole-genome sequencing data. The antibacterial enzyme had lytic activity against the biofilm of E. faecalis in vitro and in vivo. Furthermore, in aGVHD-induced gnotobiotic mice that were colonized with E. faecalis or with patient faecal samples characterized by the domination of Enterococcus, levels of intestinal cytolysin-positive E. faecalis were decreased and survival was significantly increased in the group that was treated with the E. faecalis-specific enzyme, compared with controls. Thus, administration of a phage-derived antibacterial enzyme that is specific to biofilm-forming pathogenic E. faecalis-which is difficult to eliminate with existing antibiotics-might provide an approach to protect against aGVHD.

Decreased Peripheral Blood Natural Killer Cell Count in Untreated Juvenile Dermatomyositis Is Associated with Muscle Weakness.

Juvenile Dermatomyositis (JDM) is the most common inflammatory myopathy in pediatrics. This study evaluates the role of Natural Killer (NK) cells in Juvenile Dermatomyositis (JDM) pathophysiology. The study included 133 untreated JDM children with an NK cell count evaluation before treatment. NK cell subsets (CD56low/dim vs. CD 56bright) were examined in 9 untreated children. CD56 and perforin were evaluated in situ in six untreated JDM and three orthopedic, pediatric controls. 56% of treatment-naive JDM had reduced circulating NK cell counts, designated "low NK cell". This low NK group had more active muscle disease compared to the normal NK cell group. The percentage of circulating CD56low/dim NK cells was significantly lower in the NK low group than in controls (0.55% vs. 4.6% p < 0.001). Examination of the untreated JDM diagnostic muscle biopsy documented an increased infiltration of CD56 and perforin-positive cells (p = 0.023, p = 0.038, respectively). Treatment-naive JDM with reduced circulating NK cell counts exhibited more muscle weakness and higher levels of serum muscle enzymes. Muscle biopsies from treatment-naive JDM displayed increased NK cell infiltration, with increased CD56 and perforin-positive cells.

Antigen-specific age-related memory CD8 T cells induce and track Alzheimer's-like neurodegeneration.

Cerebral (Aß) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aß/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aß/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aß/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aß/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aß/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD.

Single-Cell Analysis Reveals That CD47 mRNA Expression Correlates with Immune Cell Activation, Antiviral Isgs, and Cytotoxicity.

BACKGROUND/AIMS: Immune cells are reported to upregulate CD47 during infection, however, the role of CD47 in innate and adaptive immune cells remains unclear. METHODS: To bridge this knowledge gap, we analysed our single cell (sc)-RNA dataset along with other publicly available sc-RNA datasets from healthy controls, people with HIV-1 (PWH) and COVID-19 patients. We characterized each immune cell based on low, intermediate, and high expression of CD47 . RESULTS: Our analyses revealed that CD47 high pDCs and monocytes exhibited relatively higher expression of IFN-α regulatory genes, antiviral interferon-stimulated genes (ISGs) and MHC-I associated genes compared to CD47 inter. and CD47 low cells. Furthermore, CD47 high NK and CD8+ T cells showed higher expression of antiviral ISGs, as well as genes encoding for cytotoxic markers like granzyme B, perforin, granulysin, interferon gamma and NKG7. Additionally, CD47 high CD8+ T cells expressed higher levels of PD-1 and LAG-3 genes. Lastly, we found that CD47 high B cells had enriched expression of genes involved in cell activation and humoral responses. CONCLUSION: Overall, our analyses revealed that innate and adaptive immune cells expressing elevated activation and functional gene signatures also express higher CD47 levels.

Use of a platform with lens-free shadow imaging technology to monitor natural killer cell activity.

Natural killer (NK) cells are a crucial component of the innate immune system. This study introduces Cellytics NK, a novel platform for rapid and precise measurement of NK cell activity. This platform combines an NK-specific activation stimulator cocktail (ASC) and lens-free shadow imaging technology (LSIT), using optoelectronic components. LSIT captures digital hologram images of resting and ASC-activated NK cells, while an algorithm evaluates cell size and cytoplasmic complexity using shadow parameters. The combined shadow parameter derived from the peak-to-peak distance and width standard deviation rapidly distinguishes active NK cells from inactive NK cells at the single-cell level within 30 s. Here, the feasibility of the system was demonstrated by assessing NK cells from healthy donors and immunocompromised cancer patients, demonstrating a significant difference in the innate immunity index (I3). Cancer patients showed a lower I3 value (161%) than healthy donors (326%). I3 was strongly correlated with NK cell activity measured using various markers such as interferon-gamma, tumor necrosis factor-alpha, perforin, granzyme B, and CD107a. This technology holds promise for advancing immune functional assays, offering rapid and accurate on-site analysis of NK cells, a crucial innate immune cell, with its compact and cost-effective optoelectronic setup, especially in the post-COVID-19 era.

Acute Ascaris infection impairs the effector functions of natural killer cells in single and Salmonella co-infected pigs.

Natural killer (NK) cells play a key role in defense against Salmonella infections during the early phase of infection. Our previous work showed that the excretory/secretory products of Ascaris suum repressed NK activity in vitro. Here, we asked if NK cell functionality was influenced in domestic pigs during coinfection with Ascaris and Salmonella enterica serotype Typhimurium. Ascaris coinfection completely abolished the IL-12 and IL-18 driven elevation of IFN-γ production seen in CD16 + CD8α + perforin + NK cells of Salmonella single-infected pigs. Furthermore, Ascaris coinfection prohibited the Salmonella-driven rise in NK perforin levels and CD107a surface expression. In line with impaired effector functions, NK cells from Ascaris-single and coinfected pigs displayed elevated expression of the inhibitory KLRA1 and NKG2A receptors genes, contrasting with the higher expression of the activating NKp46 and NKp30 receptors in NK cells during Salmonella single infection. These differences were accompanied by the highly significant upregulation of T-bet protein expression in NK cells from Ascaris-single and Ascaris/Salmonella coinfected pigs. Together, our data strongly indicate a profound repression of NK functionality by an Ascaris infection which may hinder infected individuals from adequately responding to a concurrent bacterial infection.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Active juvenile systemic lupus erythematosus is associated with distinct NK cell transcriptional and phenotypic alterations.

While adaptive immune responses have been studied extensively in SLE (systemic lupus erythematosus), there is limited and contradictory evidence regarding the contribution of natural killer (NK) cells to disease pathogenesis. There is even less evidence about the role of NK cells in the more severe phenotype with juvenile-onset (J)SLE. In this study, analysis of the phenotype and function of NK cells in a large cohort of JSLE patients demonstrated that total NK cells, as well as perforin and granzyme A expressing NK cell populations, were significantly diminished in JSLE patients compared to age- and sex-matched healthy controls. The reduction in NK cell frequency was associated with increased disease activity, and transcriptomic analysis of NK populations from active and low disease activity JSLE patients versus healthy controls confirmed that disease activity was the main driver of differential NK cell gene expression. Pathway analysis of differentially expressed genes revealed an upregulation of interferon-α responses and a downregulation of exocytosis in active disease compared to healthy controls. Further gene set enrichment analysis also demonstrated an overrepresentation of the apoptosis pathway in active disease. This points to increased propensity for apoptosis as a potential factor contributing to NK cell deficiency in JSLE.

Reassessing granzyme B: unveiling perforin-independent versatility in immune responses and therapeutic potentials.

The pivotal role of Granzyme B (GzmB) in immune responses, initially tied to cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, has extended across diverse cell types and disease models. A number of studies have challenged conventional notions, revealing GzmB activity beyond apoptosis, impacting autoimmune diseases, inflammatory disorders, cancer, and neurotoxicity. Notably, the diverse functions of GzmB unfold through Perforin-dependent and Perforin-independent mechanisms, offering clinical implications and therapeutic insights. This review underscores the multifaceted roles of GzmB, spanning immunological and pathological contexts, which call for further investigations to pave the way for innovative targeted therapies.

Immune Rejection Mediated by prf1 and gzmb Affects the Colonization of Fat Greenling (Hexagrammos otakii) Spermatogonia in Heterotransplantation.

Fish germ cell transplantation holds great potential for conserving endangered species, improving cultured fish breeds, and exploring reproductive techniques. However, low transplantation efficiency is a common issue in heterotransplantation. This study transplanted fat greenling (Hexagrammos otakii) spermatogonia into the testes of spotted sea bass (Lateolabrax maculatus) to investigate factors that might affect the colonization and fixation of heterologous transplanted germ cells. Results indicated that transplanted fat greenling spermatogonia cells were successfully detected in the early transplantation phase in spotted sea bass. Their numbers gradually decreased over time, and after 10 days post-transplantation, more than 90% of the transplanted cells underwent apoptosis. Transcriptome sequencing analysis of the testes of spotted sea bass and fat greenling spermatogonia on days 1 and 10 post-transplantation revealed that this apoptosis process involved many immune-related genes and their associated signaling pathways. Acute immune rejection marker genes prf1 and gzmb were detected in the spotted sea bass testes, while immune tolerance genes lck and zap-70 were expressed in the fat greenling spermatogonia. Additionally, differential expression of prf1 and gzmb genes was screened from spotted sea bass, with experimental evidence indicating that PRF1 and GZMB protein from spotted sea bass primarily induce apoptosis in transplanted fat greenling spermatogonia via the mitochondrial apoptosis pathway, at the protein level. This suggests that the difficulties in heterotransplantation are primarily related to acute immune rejection, with PRF1 and GZMB playing significant roles.

The presence of cytotoxic CD4 and exhausted-like CD8+ T-cells is a signature of active tuberculosis.

Chronic infections induce CD4+ T-cells with cytotoxic functions (CD4 CTLs); at present, it is still unknown whether latent tuberculosis (LTB) and active tuberculosis (ATB) induce CD4 CTLs. Plasma and cells from four patient groups-uninfected contact (UC), LTB, and ATB (divided as sensitive [DS-TB]- or resistant [DR-TB]-drug)-were evaluated by flow cytometry, q-PCR, and proteomics. The data showed that ATB patients had an increased frequency of CD4+ T-cells and a decreased frequency of CD8+ T-cells. The latter displays an exhausted-like profile characterized by CD39, CD279, and TIM-3 expression. ATB had a high frequency of CD4 + perforin+ cells, suggesting a CD4 CTL profile. The expression (at the transcriptional level) of granzyme A, granzyme B, granulysin, and perforin, as well as the genes T-bet (Tbx21) and NKG2D (Klrk1), in enriched CD4+ T-cells, confirmed the cytotoxic signature of CD4+ T-cells during ATB (which was stronger in DS-TB than in DR-TB). Moreover, proteomic analysis revealed the presence of HSP70 (in DS-TB) and annexin A5 (in DR-TB), which are molecules that have been associated with favoring the CD4 CTL profile. Finally, we found that lipids from Mycobacterium tuberculosis increased the presence of CD4 CTLs in DR-TB patients. Our data suggest that ATB is characterized by exhausted-like CD8+ T-cells, which, together with a specific microenvironment, favor the presence of CD4 CTLs.

Changes in subset distribution and impaired function of circulating natural killer cells in patients with colorectal cancer.

Natural killer (NK) cells are closely associated with malignant tumor progression and metastasis. However, studies on their relevance in colorectal cancer (CRC) are limited. We aimed to comprehensively analyze the absolute counts, phenotypes, and function of circulating NK cells in patients with CRC using multiparametric flow cytometry. The distribution of NK cell subsets in the peripheral circulation of patients with CRC was significantly altered relative to the control group. This is shown by the decreased frequency and absolute count of CD56dimCD16+ NK cells with antitumor effects, contrary to the increased frequency of CD56bright NK and CD56dimCD16- NK cells with poor or ineffective antitumor effects. NK cells in patients with CRC were functionally impaired, with decreased intracellular interferon (IFN)-γ secretion and a significantly lower percentage of cell surface granzyme B and perforin expression. In addition, IFN-γ expression decreased significantly with the tumor stage progression. Based on a comprehensive analysis of the absolute counts, phenotypes, and functional markers of NK cells, we found an altered subset distribution and impaired function of circulating NK cells in patients with CRC.

KLRG1-expressing CD8+ T cells are exhausted and polyfunctional in patients with chronic hepatitis B.

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.

Disruption of IFNγ, GZMB, PRF1, or LYST Results in Reduced Suppressive Function in Human CD8+ T Cells.

An imbalance between proinflammatory and regulatory processes underlies autoimmune disease pathogenesis. We have shown that acute relapses of multiple sclerosis are characterized by a deficit in the immune suppressive ability of CD8+ T cells. These cells play an important immune regulatory role, mediated in part through cytotoxicity (perforin [PRF]/granzyme [GZM]) and IFNγ secretion. In this study, we further investigated the importance of IFNγ-, GZMB-, PRF1-, and LYST-associated pathways in CD8+ T cell-mediated suppression. Using the CRISPR-Cas9 ribonucleoprotein transfection system, we first optimized efficient gene knockout while maintaining high viability in primary bulk human CD8+ T cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry where appropriate, as well as confirmation of insertions and/or deletions at genomic target sites. We observed that the knockout of IFNγ, GZMB, PRF1, or LYST, but not the knockout of IL4 or IL5, resulted in significantly diminished in vitro suppressive ability in these cells. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T cell-mediated suppression that could be targeted for immunotherapeutic intervention.