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The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen-specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation.

Summary: The ability of cells of the immune system to acquire features such as increased longevity and enhanced secondary responses was long thought to be restricted to cells of the adaptive immune system. Natural killer (NK) cells have challenged this notion by demonstrating that they can also gain adaptive features. This has been observed in both humans and mice during infection with cytomegalovirus (CMV). The generation of adaptive NK cells requires antigen‐specific recognition of virally infected cells through stimulatory NK receptors. These receptors lack the ability to signal on their own and rather rely on adaptor molecules that contain ITAMs for driving signals. Here, we highlight our understanding of how these receptors influence the production of adaptive NK cells and propose areas in the field that merit further investigation. [ABSTRACT FROM AUTHOR]

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother's body. The trophoblast's unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy. [ABSTRACT FROM AUTHOR]

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in gd T cells - a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. gd T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific gd T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-g ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. gd T subsets were isolated using fluorescence activated cell sorting (n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and gd-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis-infected compared to the control groups for the WC1.1+ gd T cell compartment (absolute log2 FC = 1.5 and FDR Padj. = 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM, and GZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, gd T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1+ specific subset in cattle naturally infected with M. bovis. [ABSTRACT FROM AUTHOR]

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother’s body. The trophoblast’s unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in γδ T cells – a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. γδ T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturally M. bovis-infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting (n = 10–12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1+, WC1.2+, WC1- and γδ-) was used to generate transcriptomes using bulk RNA-seq (n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis-infected compared to the control groups for the WC1.1+ γδ T cell compartment (absolute log2 FC ≥ 1.5 and FDR Padj. ≤ 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors (TBX21 and EOMES), chemokine receptors (CCR5 and CCR7), granzymes (GZMA, GZMM, and GZMH) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, γδ T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1+ specific subset in cattle naturally infected with M. bovis.

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies.

One of the KIR allele, KIR3DL1*007, was associated with the progression to acquired immunodeficiency syndrome and not with the susceptibility to HIV‐1 infection in the Japanese and Indian populations, implying that KIR3DL1*007‐positive NK cells might eliminate HIV‐infected cells less effectively than NK cells bearing the other KIR3DL1 alleles or KIR3DS1 alleles. [ABSTRACT FROM AUTHOR]

Endometriosis is a chronic inflammatory disorder, characterized by the presence of endometrial cells outside the uterine cavity. An increasing number of studies correlate the immune system with endometriosis, particularly NK receptors (NKR), which have been suggested to play an essential role in the pathogenesis of the disease. This systematic review aims to enlighten the role of NKR in endometriosis. A literature search was performed independently by two reviewers, to identify studies assessing the role of NKR in endometriosis. In total, 18 studies were included. Endometriosis pathogenesis seems to be marked by the overexpression of NK inhibitor receptors (KIRS), namely, CD158a+, KIR2DL1, CD94/NKG2A, PD-1, NKB1, and EB6, and inhibiting ligands such as PD-L1, HLA-E, HLA-G, and HLA-I. Concurrently, there is a decrease in NK-activating receptors and natural cytotoxicity receptors (NCRs), such as NKp46, NKp30, and NKG2D. The immune shift from NK surveillance to NK suppression is also apparent in the greater relative number of ITIM domains compared with ITAM domains in NKRs. In conclusion, NK receptor activity seems to dictate the immunocompetency of women to clear endometriotic cells from the peritoneal cavity. Future research could explore NKRs as therapeutic targets, such as that which is now well established in cancer therapy through immunotherapy. [ABSTRACT FROM AUTHOR]

Combined antiretroviral therapy (cART) can inhibit the replication of human immunodeficiency virus type 1 (HIV-1) and reduce viral loads in the peripheral blood to undetectable levels. However, the presence of latent HIV-1 reservoirs prevents complete HIV-1 eradication. Several drugs and strategies targeting T cells are now in clinical trials, but their effectiveness in reducing viral reservoirs has been mixed. Interestingly, innate immune natural killer (NK) cells, which are promising targets for cancer therapy, also play an important role in HIV-1 infection. NK cells are a unique innate cell population with features of adaptive immunity that can regulate adaptive and innate immune cell populations; therefore, they can be exploited for HIV-1 immunotherapy and reservoir eradication. In this review, we highlight immunotherapy strategies for HIV infection that utilize the beneficial properties of NK cells. [ABSTRACT FROM AUTHOR]

Combined antiretroviral therapy (cART) can inhibit the replication of human immunodeficiency virus type 1 (HIV-1) and reduce viral loads in the peripheral blood to undetectable levels. However, the presence of latent HIV-1 reservoirs prevents complete HIV-1 eradication. Several drugs and strategies targeting T cells are now in clinical trials, but their effectiveness in reducing viral reservoirs has been mixed. Interestingly, innate immune natural killer (NK) cells, which are promising targets for cancer therapy, also play an important role in HIV-1 infection. NK cells are a unique innate cell population with features of adaptive immunity that can regulate adaptive and innate immune cell populations; therefore, they can be exploited for HIV-1 immunotherapy and reservoir eradication. In this review, we highlight immunotherapy strategies for HIV infection that utilize the beneficial properties of NK cells.

Activation of natural killer (NK) cell function is regulated by cytokines, such as IL-2, and secreted factors upregulated in the tumor microenvironment, such as platelet-derived growth factor D (PDGF-DD). In order to elucidate a clinical role for these important regulators of NK cell function in antitumor immunity, we generated transcriptional signatures representing resting, IL-2-expanded, and PDGF-DD-activated, NK cell phenotypes and established their abundance in The Cancer Genome Atlas bladder cancer (BLCA) dataset using CIBERSORT. The IL-2-expanded NK cell phenotype was the most abundant in low and high grades of BLCA tumors and was associated with improved prognosis. In contrast, PDGFD expression was associated with numerous cancer hallmark pathways in BLCA tumors compared with normal bladder tissue, and a high tumor abundance of PDGFD transcripts and the PDGF-DD-activated NK cell phenotype were associated with a poor BLCA prognosis. Finally, high tumor expression of transcripts encoding the activating NK cell receptors, KLRK1 and the CD160–TNFRSF14 receptor–ligand pair, was strongly correlated with the IL-2-expanded NK cell phenotype and improved BLCA prognosis. The transcriptional parameters we describe may be optimized to improve BLCA patient prognosis and risk stratification in the clinic and potentially provide gene targets of therapeutic significance for enhancing NK cell antitumor immunity in BLCA. [ABSTRACT FROM AUTHOR]

Activation of natural killer (NK) cell function is regulated by cytokines, such as IL-2, and secreted factors upregulated in the tumor microenvironment, such as platelet-derived growth factor D (PDGF-DD). In order to elucidate a clinical role for these important regulators of NK cell function in antitumor immunity, we generated transcriptional signatures representing resting, IL-2-expanded, and PDGF-DD-activated, NK cell phenotypes and established their abundance in The Cancer Genome Atlas bladder cancer (BLCA) dataset using CIBERSORT. The IL-2-expanded NK cell phenotype was the most abundant in low and high grades of BLCA tumors and was associated with improved prognosis. In contrast, PDGFD expression was associated with numerous cancer hallmark pathways in BLCA tumors compared with normal bladder tissue, and a high tumor abundance of PDGFD transcripts and the PDGF-DD-activated NK cell phenotype were associated with a poor BLCA prognosis. Finally, high tumor expression of transcripts encoding the activating NK cell receptors, KLRK1 and the CD160–TNFRSF14 receptor–ligand pair, was strongly correlated with the IL-2-expanded NK cell phenotype and improved BLCA prognosis. The transcriptional parameters we describe may be optimized to improve BLCA patient prognosis and risk stratification in the clinic and potentially provide gene targets of therapeutic significance for enhancing NK cell antitumor immunity in BLCA.

Endometriosis is a chronic inflammatory disorder, characterized by the presence of endometrial cells outside the uterine cavity. An increasing number of studies correlate the immune system with endometriosis, particularly NK receptors (NKR), which have been suggested to play an essential role in the pathogenesis of the disease. This systematic review aims to enlighten the role of NKR in endometriosis. A literature search was performed independently by two reviewers, to identify studies assessing the role of NKR in endometriosis. In total, 18 studies were included. Endometriosis pathogenesis seems to be marked by the overexpression of NK inhibitor receptors (KIRS), namely, CD158a+, KIR2DL1, CD94/NKG2A, PD-1, NKB1, and EB6, and inhibiting ligands such as PD-L1, HLA-E, HLA-G, and HLA-I. Concurrently, there is a decrease in NK-activating receptors and natural cytotoxicity receptors (NCRs), such as NKp46, NKp30, and NKG2D. The immune shift from NK surveillance to NK suppression is also apparent in the greater relative number of ITIM domains compared with ITAM domains in NKRs. In conclusion, NK receptor activity seems to dictate the immunocompetency of women to clear endometriotic cells from the peritoneal cavity. Future research could explore NKRs as therapeutic targets, such as that which is now well established in cancer therapy through immunotherapy.

The objective of the study is to estimate the potential of gintonin, as an immune enhancing agent through natural killer cell (NK cell) activity in cyclophosphamide (CY)-induced immunosuppressive animals. Accumulated results reveals that, gintonin attenuated CY-induced immunosuppression and it might modulate NK cell activity to boost the immunity., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper., (© 2024 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies.

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies. [ABSTRACT FROM AUTHOR]

The NK cells functions are finely tuned by several kinds of inhibitory and activating receptors, whose pattern of expression characterizes different NK subpopulations and varies with the cell activation status. MicroRNAs have an important role in tightly regulating the expression of NK receptors and, analogously, the expression of their ligands in target cells. The relevance of the microRNA-mediated control is highlighted by the dysregulation of these pathways observed in cancer and virus-infected cells. Here we review our current knowledge of the microRNAs involved in the regulation of NK receptors, as well as that of the corresponding cellular ligands.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis , continues to cause significant issues for the global agriculture industry as well as for human health. An incomplete understanding of the host immune response contributes to the challenges of control and eradication of this zoonotic disease. In this study, high-throughput bulk RNA sequencing (RNA-seq) was used to characterise differential gene expression in γδ T cells - a subgroup of T cells that bridge innate and adaptive immunity and have known anti-mycobacterial response mechanisms. γδ T cell subsets are classified based on expression of a pathogen-recognition receptor known as Workshop Cluster 1 (WC1) and we hypothesised that bTB disease may alter the phenotype and function of specific γδ T cell subsets. Peripheral blood was collected from naturally M. bovis -infected (positive for single intradermal comparative tuberculin test (SICTT) and IFN-γ ELISA) and age- and sex-matched, non-infected control Holstein-Friesian cattle. γδ T subsets were isolated using fluorescence activated cell sorting ( n = 10-12 per group) and high-quality RNA extracted from each purified lymphocyte subset (WC1.1 + , WC1.2 + , WC1 - and γδ - ) was used to generate transcriptomes using bulk RNA-seq ( n = 6 per group, representing a total of 48 RNA-seq libraries). Relatively low numbers of differentially expressed genes (DEGs) were observed between most cell subsets; however, 189 genes were significantly differentially expressed in the M. bovis -infected compared to the control groups for the WC1.1 + γδ T cell compartment (absolute log 2 FC ≥ 1.5 and FDR P adj. ≤ 0.1). The majority of these DEGs (168) were significantly increased in expression in cells from the bTB+ cattle and included genes encoding transcription factors ( TBX21 and EOMES ), chemokine receptors ( CCR5 and CCR7 ), granzymes ( GZMA, GZMM , and GZMH ) and multiple killer cell immunoglobulin-like receptor (KIR) proteins indicating cytotoxic functions. Biological pathway overrepresentation analysis revealed enrichment of genes with multiple immune functions including cell activation, proliferation, chemotaxis, and cytotoxicity of lymphocytes. In conclusion, γδ T cells have important inflammatory and regulatory functions in cattle, and we provide evidence for preferential differential activation of the WC1.1 + specific subset in cattle naturally infected with M. bovis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Bhat, Elnaggar, Hall, McHugo, Reid, MacHugh and Meade.)

Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deep molecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56bright and CD56dim NK cell subsets and the expression of CD107a and IFN-γ in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35–65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation.

X-linked lymphoproliferative disease 1 (XLP1) is a monogenic disorder caused by mutations in SH2D1A , resulting in the absence/dysfunction of the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). Consequently, SLAM receptors as 2B4 (CD244) and NTB-A (SLAMF6), upon ligand engagement, exert inhibitory instead of activating function. This causes an immune dysfunction that is worsened by the selective inability of NK and T cells to kill EBV-infected B cells with dramatic clinical sequelae (e.g. fulminant mononucleosis, hyperinflammation, lymphoma). Here we outline recent findings on the interplay between inhibitory 2B4 and the various activating receptors in NK cells. 2B4 engagement selectively blocks ITAM-dependent activating receptors as NCR and CD16, while it does not affect NKG2D and DNAM-1. Furthermore, inhibitory 2B4 participates to NK cell education, as highlighted by the existence in XLP1 patients of a large subset of fully functional NK cells that lack self-HLA specific inhibitory receptors and exert autoreactivity against mature dendritic cells. [ABSTRACT FROM AUTHOR]

The last decade will be remembered as the dawn of the immunotherapy era during which we have witnessed the approval by regulatory agencies of genetically engineered CAR T-cells and of checkpoint inhibitors for cancer treatment. Understandably, T-lymphocytes represent the essential player in these approaches. These cells can mediate impressive tumor regression in terminally-ill cancer patients. Moreover, they are amenable to genetic engineering to improve their function and specificity. In the present review, we will give an overview of the most recent developments in the field of T-cell genetic engineering including TCR-gene transfer and CAR T-cells strategies. We will also elaborate on the development of other types of genetic modifications to enhance their anti-tumor immune response such as the use of co-stimulatory chimeric receptors (CCRs) and unconventional CARs built on non-antibody molecules. Finally, we will discuss recent advances in genome editing and synthetic biology applied to T-cell engineering and comment on the next challenges ahead. T-cells isolated from the patient are activated ex vivo and modified genetically (e.g. , using a viral vector) to express new gene(s) that will endow the cell with target specificity and/or improved immunological function. Unlabelled Image [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deep molecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56bright and CD56dim NK cell subsets and the expression of CD107a and IFN-γ in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35–65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation. [ABSTRACT FROM AUTHOR]

Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens.

The administration of ex vivo-expanded Natural Killer (NK) cells in leukemia therapy is still challenging, in part due to the difficulty to generate in sufficient quantities fully mature and functional NK cells and Identification of surface markers indicative of NK maturation and functionality is therefore needed. Here, based on the analysis of surface receptors of ex vivo-expanded NK cells, we identified CD94 as a surface marker correlating with high lytic potential against leukemic cell lines and immunological synapse formation. CD94-positive ex vivo-expanded NK cells displayed higher expression of NKG2 receptors and the adhesion molecule LFA-1, as compared with their CD94-negative counterparts. We also tested the in vivo anti-leukemic capacity of ex vivo-expanded NK cells against patient-derived acute myeloid leukemia cells. Although no anti-leukemic effect was detected, we noticed that only CD94-positive ex vivo-expanded NK cells were detected in leukemic mice at the end of the 2-week treatment. Moreover, flow cytometry analysis showed a subpopulation harboring CD94 (NK) and CD34 (leukemic cells) double staining, indicative of conjugate formation. Therefore surface expression of CD94 on ex vivo-differentiated NK cells emerged as an indicator of in vitro and in vivo killer cell functionality.

Keywords: conventional NK cells; tissue resident NK cells; innate lymphoid cells; tissue microenvironment; NK receptors As described in detail by Mikulak et al., human liver contains three NK cell populations showing transcriptional and phenotypic differences: liver tr-NK cells, memory-like NK cells and recirculating c-NK cells. Although this issue could be the subject of a dedicated Research Topic, we here present a few contributions highlighting two specific components when considering any NK cell-based immunotherapeutic strategy: NK cell checkpoints and tumor infiltration by NK cells. Conventional NK cells, tissue resident NK cells, innate lymphoid cells, tissue microenvironment, NK receptors. [Extracted from the article]

Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens. [ABSTRACT FROM AUTHOR]

Aging is associated with an increased susceptibility to infection and a failure to control latent viruses thought to be driven, at least in part, by alterations in CD8 T cell function. The aging T cell repertoire is characterized by an accumulation of effector CD8 T cells, many of which express the negative regulatory receptor CD85j. To define the biological significance of CD85j expression on CD8 T cells and to address the question whether presence of CD85j in older individuals is beneficial or detrimental for immune function, we examined the specific attributes of CD8 T cells expressing CD85j as well as the functional role of CD85j in antigen-specific CD8 T cell responses during immune aging. Here, we show that CD85j is mainly expressed by terminally differentiated effector (TEMRAs) CD8 T cells, which increase with age, in cytomegalovirus (CMV) infection and in males. CD85j+ CMV-specific cells demonstrate clonal expansion. However, TCR diversity is similar between CD85j+ and CD85j− compartments, suggesting that CD85j does not directly impact the repertoire of antigen-specific cells. Further phenotypic and functional analyses revealed that CD85j identifies a specific subset of CMV-responsive CD8 T cells that coexpress a marker of senescence (CD57) but retain polyfunctional cytokine production and expression of cytotoxic mediators. Blocking CD85j binding enhanced proliferation of CMV-specific CD8 T cells upon antigen stimulation but did not alter polyfunctional cytokine production. Taken together, these data demonstrate that CD85j characterizes a population of “senescent,” but not exhausted antigen-specific effector CD8 T cells and indicates that CD85j is an important checkpoint regulator controlling expansion of virus-specific T cells during aging. Inhibition of CD85j activity may be a mechanism to promote stronger CD8 T cell effector responses during immune aging.

Aging is associated with an increased susceptibility to infection and a failure to control latent viruses thought to be driven, at least in part, by alterations in CD8 T cell function. The aging T cell repertoire is characterized by an accumulation of effector CD8 T cells, many of which express the negative regulatory receptor CD85j. To define the biological significance of CD85j expression on CD8 T cells and to address the question whether presence of CD85j in older individuals is beneficial or detrimental for immune function, we examined the specific attributes of CD8 T cells expressing CD85j as well as the functional role of CD85j in antigen-specific CD8 T cell responses during immune aging. Here, we show that CD85j is mainly expressed by terminally differentiated effector (TEMRAs) CD8 T cells, which increase with age, in cytomegalovirus (CMV) infection and in males. CD85j+ CMV-specific cells demonstrate clonal expansion. However, TCR diversity is similar between CD85j+ and CD85j- compartments, suggesting that CD85j does not directly impact the repertoire of antigen-specific cells. Further phenotypic and functional analyses revealed that CD85j identifies a specific subset of CMV-responsive CD8 T cells that coexpress a marker of senescence (CD57) but retain polyfunctional cytokine production and expression of cytotoxic mediators. Blocking CD85j binding enhanced proliferation of CMV-specific CD8 T cells upon antigen stimulation but did not alter polyfunctional cytokine production. Taken together, these data demonstrate that CD85j characterizes a population of "senescent," but not exhausted antigen-specific effector CD8 T cells and indicates that CD85j is an important checkpoint regulator controlling expansion of virus-specific T cells during aging. Inhibition of CD85j activity may be a mechanism to promote stronger CD8 T cell effector responses during immune aging. [ABSTRACT FROM AUTHOR]

X-linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)-associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV-infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig-like receptor (KIR)/KIR-L genotype and phenotypic repertoire of self-HLA class I specific inhibitory NK receptors (self-iNKRs). We also analyzed NK-cell cytotoxicity against CD48+ or CD48− KIR-ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self-iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48+ EBV-transformed B cells and M1 macrophages. Importantly, self-iNKR defective NK cells kill CD48− targets, such as mature DCs. Self-iNKR− NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK-cell maturation. Killing of autologous mature DC by self-iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients' immune defect. [ABSTRACT FROM AUTHOR]

Background/aim: We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells., Materials and Methods: γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo., Results: The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1., Conclusion: The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines ( n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS. [ABSTRACT FROM AUTHOR]

Endometriosis is a chronic inflammatory disorder, characterized by the presence of endometrial cells outside the uterine cavity. An increasing number of studies correlate the immune system with endometriosis, particularly NK receptors (NKR), which have been suggested to play an essential role in the pathogenesis of the disease. This systematic review aims to enlighten the role of NKR in endometriosis. A literature search was performed independently by two reviewers, to identify studies assessing the role of NKR in endometriosis. In total, 18 studies were included. Endometriosis pathogenesis seems to be marked by the overexpression of NK inhibitor receptors (KIRS), namely, CD158a+, KIR2DL1, CD94/NKG2A, PD-1, NKB1, and EB6, and inhibiting ligands such as PD-L1, HLA-E, HLA-G, and HLA-I. Concurrently, there is a decrease in NK-activating receptors and natural cytotoxicity receptors (NCRs), such as NKp46, NKp30, and NKG2D. The immune shift from NK surveillance to NK suppression is also apparent in the greater relative number of ITIM domains compared with ITAM domains in NKRs. In conclusion, NK receptor activity seems to dictate the immunocompetency of women to clear endometriotic cells from the peritoneal cavity. Future research could explore NKRs as therapeutic targets, such as that which is now well established in cancer therapy through immunotherapy.

Invariant natural killer T (iNKT) cells to date represent the best example of cells known to have a hybrid function, representing both innate and adaptive immunity. Shared phenotypic similarities with NK cells together with a rapid response to a cytokine stimulus and a productive TCR engagement are the features that underline the hybrid nature of iNKT cells. Using these criteria, we provide molecular and functional evidence demonstrating that CD1d-inde-pendent (CD1dind) NKT cells, a population of CD1d-unrestricted NKT cells, are endowed with a hybrid function far superior to that of iNKT cells: (i) an extensive shared program with NK cells, (ii) a closer Euclidian distance with NK cells, and (iii) the ability to respond to innate stimuli (Poly:IC) with cytotoxic potential in the same manner as NK cells identify a hybrid feature in CD1dindNKT cells that truly fulfills the dual function of an NK and a T cell. Our finding that CD1dindNKT cells are programmed to act like NK cells in response to innate signals while being capable of adaptive responses is unprecedented, and thus might reemphasize CD1d-unrestricted NKT cells as a subset of lymphocytes that could affect biological processes of antimicrobial and tumor immunity in a unique way. [ABSTRACT FROM AUTHOR]

Abstract: The study of in vitro and in vivo NK cell differentiation from hematopoietic precursors revealed the existence of discrete stages of development. These stages are characterized by the progressive acquisition of markers and receptors that play a crucial role in NK cell function. The knowledge acquired has revealed particularly relevant for improving the HSCT to cure high-risk leukemias in the haplo-HSCT setting, in which NK cells play a central role in the clearance of leukemic cells and in the positive clinical outcome. [Copyright &y& Elsevier]

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner. [ABSTRACT FROM AUTHOR]

The MHC fold is found in proteins that have a range of functions in the maintenance of an organism's health, from immune regulation to fat metabolism. Well adapted for antigen presentation, as seen for peptides in the classical MHC molecules and for lipids in CD 1 molecules, the MHC fold has also been modified to perform Fc-receptor activity (e.g., FcRn) and for roles in host homeostasis (e.g., with HFE and ZAG). The more divergent MHC-like molecules, such as some of those that interact with the NKG2D receptor, represent the minimal MHC fold, doing away with the α3 domain and β while maintaining the αl/α2 platform domain for receptor engagement. Viruses have also co-opted the MHC fold for immune-evasive functions. The variations on the theme of a (3-sheet topped by two semiparallel a-helices are discussed in this review, highlighting the fantastic adaptability of this fold for good and for bad. [ABSTRACT FROM AUTHOR]

Despite recent advances with monoclonal antibody therapy, chronic lymphocytic leukemia (CLL) remains incurable. Natural killer (NK) cells are potent antitumoral effectors, particularly against hematological malignancies. Defective recognition of B-CLL leukemic cells by NK cells has been previously described. Here, we deciphered the mechanisms that hamper NK cell-mediated clearance of B-CLL and evaluated the potential of NK cells as therapeutic tools for treatment of CLL. First of all, leukemic B cells resemble to normal B cells with a weak expression of ligands for NK receptors. Conversely, NK cells from B-CLL patients were functionally and phenotypically competent, despite a decrease of expression of the activating receptor NKp30. Consequently, resting allogeneic NK cells were unable to kill leukemic B cells in vitro. These data suggest that patients' NK cells cannot initiate a proper immune reaction due to a lack of leukemic cell recognition. We next set up a xenotransplantation mouse model to study NK-CLL cell interactions. Together with our in vitro studies, in vivo data revealed that activation of NK cells is required in order to control B-CLL and that activated NK cells synergize to enhance rituximab effect on tumor load. This study points out the requirements for immune system manipulation for treatment of B-CLL in combination with monoclonal antibody therapy. [ABSTRACT FROM AUTHOR]