Early CAR- CD4+ T-lymphocytes recovery following CAR-T cell infusion: A worse outcome in diffuse large B cell lymphoma.

CAR- CD4+ T cell lymphopenia is an emerging issue following CAR-T cell therapy. We analyzed the determinants of CD4+ T cell recovery and a possible association with survival in 31 consecutive patients treated with commercial CAR-T for diffuse large B-cell (DLBCL) or mantle cell lymphoma. Circulating immune subpopulations were characterized through multiparametric-flow cytometry. Six-month cumulative incidence of CAR- CD4+ T cell recovery (≥200 cells/µL) was 0.43 (95% confidence interval [CI]: 0.28-0.65). Among possible determinants of CD4+ T cell recovery, we recognized infusion of a 4-1BB product (tisagenlecleucel, TSA) in comparison with a CD28 (axicabtagene/brexucabtagene, AXI/BRX) (hazard ratio [HR] [95% CI]: 5.79 [1.16-24.12] p = 0.016). Higher CD4+ T cell counts resulted with TSA at month-1, -2 and -3. Moderate-to-severe infections were registered with prolonged CD4+ T cell lymphopenia. Early, month-1 CD4+ T cell recovery was associated with a worse outcome in the DLBCL cohort, upheld in a multivariate regression model for overall survival (HR: 4.46 [95% CI: 1.12-17.71], p = 0.03). We conclude that a faster CAR- CD4+ T cell recovery is associated with TSA as compared to AXI/BRX. Month-1 CAR- CD4+ T cell subset recovery could represent a "red flag" for CAR-T cell therapy failure in DLBCL patients.

Identification of a novel cord blood NK cell subpopulation expressing functional programmed death receptor-1.

Background: Natural Killer cells (NKs) represent the innate counterpart of TCRαß lymphocytes and are characterized by a high anti-tumor and an anti-viral cytotoxic activity. Recently, it has been demonstrated that NKs can express PD-1 as an additional inhibitory receptor. Specifically, PD-1 was identified on a subpopulation of terminally differentiated NKs from healthy adults with previous HCMV infection. So far it is unknown whether PD-1 appears during NK-cell development and whether this process is directly or indirectly related to HCMV infection. Methods: In this study, we analyzed the expression and function of PD-1 on Cord Blood derived NKs (CB-NKs) on a large cohort of newborns through multiparametric cytofluorimetric analysis. Results: We identified PD-1 on CB-NKs in more than of half the newborns analyzed. PD-1 was present on CD56dim NKs, and particularly abundant on CD56neg NKs, but only rarely present on CD56bright NKs. Importantly, unlike in adult healthy donors, in CB-NKs PD-1 is co-expressed not only with KIR, but also with NKG2A. PD-1 expression was independent of HCMV mother seropositivity and occurs in the absence of HCMV infection/reactivation during pregnancy. Notably, PD-1 expressed on CB-NKs was functional and mediated negative signals when triggered. Conclusion: To our understanding, this study is the first to report PD-1 expression on CB derived NKs and its features in perinatal conditions. These data may prove important in selecting the most suitable CB derived NK cell population for the development of different immunotherapeutic treatments.

NK Cell-Based Immunotherapy in Colorectal Cancer.

Human Natural Killer (NK) cells are all round players in immunity thanks to their powerful and immediate response against transformed cells and the ability to modulate the subsequent adaptive immune response. The potential of immunotherapies based on NK cell involvement has been initially revealed in the hematological setting but has inspired the design of different immune tools to also be applied against solid tumors, including colorectal cancer (CRC). Indeed, despite cancer prevention screening plans, surgery, and chemotherapy strategies, CRC is one of the most widespread cancers and with the highest mortality rate. Therefore, further efficient and complementary immune-based therapies are in urgent need. In this review, we gathered the most recent advances in NK cell-based immunotherapies aimed at fighting CRC, in particular, the use of monoclonal antibodies targeting tumor-associated antigens (TAAs), immune checkpoint blockade, and adoptive NK cell therapy, including NK cells modified with chimeric antigen receptor (CAR-NK).

CD19-Targeted Immunotherapies for Diffuse Large B-Cell Lymphoma.

Surgical resection, chemotherapy and radiotherapy were, for many years, the only available cancer treatments. Recently, the use of immune checkpoint inhibitors and adoptive cell therapies has emerged as promising alternative. These cancer immunotherapies are aimed to support or harness the patient's immune system to recognize and destroy cancer cells. Preclinical and clinical studies, based on the use of T cells and more recently NK cells genetically modified with chimeric antigen receptors retargeting the adoptive cell therapy towards tumor cells, have already shown remarkable results. In this review, we outline the latest highlights and progress in immunotherapies for the treatment of Diffuse Large B-cell Lymphoma (DLBCL) patients, focusing on CD19-targeted immunotherapies. We also discuss current clinical trials and opportunities of using immunotherapies to treat DLBCL patients.

Role of the Main Non HLA-Specific Activating NK Receptors in Pancreatic, Colorectal and Gastric Tumors Surveillance.

Human NK cells can control tumor growth and metastatic spread thanks to their powerful cytolytic activity which relies on the expression of an array of activating receptors. Natural cytotoxicity receptors (NCRs) NKG2D and DNAM-1 are those non-HLA-specific activating NK receptors that are mainly involved in sensing tumor transformation by the recognition of different ligands, often stress-induced molecules, on the surface of cancer cells. Tumors display several mechanisms aimed at dampening/evading NK-mediated responses, a relevant fraction of which is based on the downregulation of the expression of activating receptors and/or their ligands. In this review, we summarize the role of the main non-HLA-specific activating NK receptors, NCRs, NKG2D and DNAM-1, in controlling tumor growth and metastatic spread in solid malignancies affecting the gastrointestinal tract with high incidence in the world population, i.e., pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and gastric cancer (GC), also describing the phenotypic and functional alterations induced on NK cells by their tumor microenvironment.

NK cells as adoptive cellular therapy for hematological malignancies: Advantages and hurdles.

Natural killer cells are an essential component of the innate immune system and play a crucial role in immunity against malignancies, without, at difference with T cells, requiring antigen priming or inducing graft-versus-host-disease. Hence, Natural Killer cells can provide a valuable source of allogeneic "off-the-shelf" adoptive therapy and mediate major antileukemia effects, without inducing potentially lethal alloreactivity. Several cell sources have been used for producing and expanding large numbers of clinical-grade natural killer cells. In this review, we will discuss the advantages and challenges of Natural Killer cell-based therapeutic approaches for hematological malignancies, also exploring different strategies to potentiate their clinical application.

Inhibitory Receptors and Checkpoints in Human NK Cells, Implications for the Immunotherapy of Cancer.

The highly destructive mechanisms by which the immune system faces microbial infections is under the control of a series of inhibitory receptors. While most of these receptors prevent unwanted/excessive responses of individual effector cells, others play a more general role in immunity, acting as true inhibitory checkpoints controlling both innate and adaptive immunity. Regarding human NK cells, their function is finely regulated by HLA-class I-specific inhibitory receptors which allow discrimination between HLA-I+, healthy cells and tumor or virus-infected cells displaying loss or substantial alterations of HLA-I molecules, including allelic losses that are sensed by KIRs. A number of non-HLA-specific receptors have been identified which recognize cell surface or extracellular matrix ligands and may contribute to the physiologic control of immune responses and tolerance. Among these receptors, Siglec 7 (p75/AIRM-1), LAIR-1 and IRp60, recognize ligands including sialic acids, extracellular matrix/collagen or aminophospholipids, respectively. These ligands may be expressed at the surface of tumor cells, thus inhibiting NK cell function. Expression of the PD-1 checkpoint by NK cells requires particular cytokines (IL-15, IL-12, IL-18) together with cortisol, a combination that may occur in the microenvironment of different tumors. Blocking of single or combinations of inhibitory receptors unleashes NK cells and restore their anti-tumor activity, with obvious implications for tumor immunotherapy.

Natural killer cell impairment in ovarian clear cell carcinoma.

In the present study, we report the analysis of NK cells derived from patients suffering from a rare ovarian cancer histotype of clear cell carcinoma (OCCC) resistant to conventional chemotherapies. We analyzed the phenotype of NK cells derived from peripheral blood (PB) and peritoneal fluid (PF) and evaluated cytotoxic interactions between NK cells and autologous tumor cells (ATC) derived from patients. We provided evidence of impaired degranulation capacity of NK cells derived from patients' PF in the presence of ATC. Analyzing tumor cell ligands recognized by NK cell receptors, we found that ATC are characterized by an HLA class I+ phenotype (although the level of HLA-I expression varies among all patients) and by a heterogeneous expression of ligands for activating NK receptors (from normal to decreased expression of some markers). Furthermore, we observed a down-regulation of crucial NK cell activating receptors, primarily DNAX Accessory Molecule-1 (DNAM-1), on tumor-associated NK cells. Based on these results, we propose that this severe lysis defect may be due to both negative interactions between HLA-I-specific inhibitory NK cell receptors/HLA-I molecules and to defective interactions between activating NK receptors and cognate ligands. In conclusion, for the first time, the phenotypic and functional properties of tumor-associated NK cells and their ATC derived from PF of patients with advanced stage of OCCC were characterized. Taken together results indicate altered interactions between NK cells and ATC and shed light on the aggressive mechanisms of this cancer histotype. Further studies on this rare tumor will be helpful to improve and define more effective therapies.

miRNAs in NK Cell-Based Immune Responses and Cancer Immunotherapy.

The incidence of certain forms of tumors has increased progressively in recent years and is expected to continue growing as life expectancy continues to increase. Tumor-infiltrating NK cells may contribute to develop an anti-tumor response. Optimized combinations of different cancer therapies, including NK cell-based approaches for targeting tumor cells, have the potential to open new avenues in cancer immunotherapy. Functional inhibitory receptors on NK cells are needed to prevent their attack on healthy cells. Nevertheless, disruption of inhibitory receptors function on NK cells increases the cytotoxic capacity of NK cells against cancer cells. MicroRNAs (miRNAs) are small non-coding RNA molecules that target mRNA and thus regulate the expression of genes involved in the development, maturation, and effector functions of NK cells. Therapeutic strategies that target the regulatory effects of miRNAs have the potential to improve the efficiency of cancer immunotherapy. Interestingly, emerging evidence points out that some miRNAs can, directly and indirectly, control the surface expression of immune checkpoints on NK cells or that of their ligands on tumor cells. This suggests a possible use of miRNAs in the context of anti-tumor therapy. This review provides the current overview of the connections between miRNAs and regulation of NK cell functions and discusses the potential of these miRNAs as innovative biomarkers/targets for cancer immunotherapy.

NK Cell-Based Immunotherapy for Hematological Malignancies.

Natural killer (NK) lymphocytes are an integral component of the innate immune system and represent important effector cells in cancer immunotherapy, particularly in the control of hematological malignancies. Refined knowledge of NK cellular and molecular biology has fueled the interest in NK cell-based antitumor therapies, and recent efforts have been made to exploit the high potential of these cells in clinical practice. Infusion of high numbers of mature NK cells through the novel graft manipulation based on the selective depletion of T cells and CD19+ B cells has resulted into an improved outcome in children with acute leukemia given human leucocyte antigen (HLA)-haploidentical hematopoietic transplantation. Likewise, adoptive transfer of purified third-party NK cells showed promising results in patients with myeloid malignancies. Strategies based on the use of cytokines or monoclonal antibodies able to induce and optimize NK cell activation, persistence, and expansion also represent a novel field of investigation with remarkable perspectives of favorably impacting on outcome of patients with hematological neoplasia. In addition, preliminary results suggest that engineering of mature NK cells through chimeric antigen receptor (CAR) constructs deserve further investigation, with the goal of obtaining an "off-the-shelf" NK cell bank that may serve many different recipients for granting an efficient antileukemia activity.

Different Features of Tumor-Associated NK Cells in Patients With Low-Grade or High-Grade Peritoneal Carcinomatosis.

Peritoneal carcinomatosis (PC) is a rare disease defined as diffused implantation of neoplastic cells in the peritoneal cavity. This clinical picture occurs during the evolution of peritoneal tumors, and it is the main cause of morbidity and mortality of patients affected by these pathologies, though cytoreductive surgery with heated intra-peritoneal chemotherapy (CRS/HIPEC) is yielding promising results. In the present study, we evaluated whether the tumor microenvironment of low-grade and high-grade PC could affect the phenotypic and functional features and thus the anti-tumor potential of NK cells. We show that while in the peritoneal fluid (PF) of low-grade PC most CD56dim NK cells show a relatively immature phenotype (NKG2A+KIR-CD57-CD16dim), in the PF of high-grade PC NK cells are, in large majority, mature (CD56dimKIR+CD57+CD16bright). Furthermore, in low-grade PC, PF-NK cells are characterized by a sharp down-regulation of some activating receptors, primarily NKp30 and DNAM-1, while, in high-grade PC, PF-NK cells display a higher expression of the PD-1 inhibitory checkpoint. The compromised phenotype observed in low-grade PC patients corresponds to a functional impairment. On the other hand, in the high-grade PC patients PF-NK cells show much more important defects that only partially reflect the compromised phenotype detected. These data suggest that the PC microenvironment may contribute to tumor escape from immune surveillance by inducing different NK cell impaired features leading to altered anti-tumor activity. Notably, after CRS/HIPEC treatment, the altered NK cell phenotype of a patient with a low-grade disease and favorable prognosis was reverted to a normal one. Our present data offer a clue for the development of new immunotherapeutic strategies capable of restoring the NK-mediated anti-tumor responses in association with the CRS/HIPEC treatment to increase the effectiveness of the current therapy.

An Historical Overview: The Discovery of How NK Cells Can Kill Enemies, Recruit Defense Troops, and More.

Natural killer (NK) cells were originally defined as effector lymphocytes of innate immunity characterized by the unique ability of killing tumor and virally infected cells without any prior priming and expansion of specific clones. The "missing-self" theory, proposed by Klas Karre, the seminal discovery of the first prototypic HLA class I-specific inhibitory receptors, and, later, of the Natural Cytotoxicity Receptors (NCRs) by Alessandro Moretta, provided the bases to understand the puzzling behavior of NK cells. Actually, those discoveries proved crucial also for many of the achievements that, along the years, have contributed to the modern view of these cells. Indeed, NK cells, besides killing susceptible targets, are now known to functionally interact with different immune cells, sense pathogens using TLR, adapt their responses to the local environment, and, even, mount a sort of immunological memory. In this review, we will specifically focus on the main activating NK receptors and on their crucial role in the ever-increasing number of functions assigned to NK cells and other innate lymphoid cells (ILCs).

Features of Memory-Like and PD-1(+) Human NK Cell Subsets.

Human NK cells are distinguished into CD56(bright)CD16(-) cells and CD56(dim)CD16(+) cells. These two subsets are conventionally associated with differential functional outcomes and are heterogeneous with respect to the expression of KIR and CD94/NKG2 heterodimers that represent the two major types of HLA-class I-specific receptors. Recent studies indicated that immature CD56(bright) NK cells, homogeneously expressing the inhibitory CD94/NKG2A receptor, are precursors of CD56(dim) NK cells that, in turn, during their process of differentiation, lose expression of CD94/NKG2A and subsequentially acquire inhibitory KIRs and LIR-1. The terminally differentiated phenotype of CD56(dim) cells is marked by the expression of the CD57 molecule that is associated with poor responsiveness to cytokine stimulation, but retained cytolytic capacity. Remarkably, this NKG2A(-)KIR(+)LIR-1(+)CD57(+)CD56(dim) NK cell subset when derived from individuals previously exposed to pathogens, such as human cytomegalovirus (HCMV), may contain "memory-like" NK cells. These cells are generally characterized by an upregulation of the activating receptor CD94/NKG2C and a downregulation of the inhibitory receptor Siglec-7. The "memory-like" NK cells are persistent over time and display some hallmarks of adaptive immunity, i.e., clonal expansion, more effective antitumor and antiviral immune responses, longevity, as well as given epigenetic modifications. Interestingly, unknown cofactors associated with HCMV infection may induce the onset of a recently identified fully mature NK cell subset, characterized by marked downregulation of the activating receptors NKp30 and NKp46 and by the unexpected expression of the inhibitory PD-1 receptor. This phenotype correlates with an impaired antitumor NK cell activity that can be partially restored by antibody-mediated disruption of PD-1/PD-L interaction.

Activating KIRs and NKG2C in Viral Infections: Toward NK Cell Memory?

Natural killer (NK) cells are important players in the immune defense against viral infections. The contribution of activating killer immunoglobulin-like receptors (KIRs) and CD94/NKG2C in regulating anti-viral responses has recently emerged. Thus, in the hematopoietic stem cell transplantation setting, the presence of donor activating KIRs (aKIRs) may protect against viral infections, while in HIV-infected individuals, KIR3DS1, in combination with HLA-Bw4-I80, results in reduction of viral progression. Since, studies have been performed mainly at the genetic or transcriptional level, the effective size, the function, and the "licensing" status of NK cells expressing aKIRs, as well as the nature of their viral ligands, require further investigation. Certain viral infections, mainly due to Human cytomegalovirus (HCMV), can deeply influence the NK cell development and function by inducing a marked expansion of mature NKG2C(+) NK cells expressing self-activating KIRs. This suggests that NKG2C and/or aKIRs are involved in the selective proliferation of this subset. The persistent, HCMV-induced, imprinting suggests that NK cells may display unexpected adaptive immune traits. The role of aKIRs and NKG2C in regulating NK cell responses and promoting a memory-like response to certain viruses is discussed.

Natural Killer (NK)/melanoma cell interaction induces NK-mediated release of chemotactic High Mobility Group Box-1 (HMGB1) capable of amplifying NK cell recruitment.

In this study we characterize a new mechanism by which Natural Killer (NK) cells may amplify their recruitment to tumors. We show that NK cells, upon interaction with melanoma cells, can release a chemotactic form of High Mobility Group Box-1 (HMGB1) protein capable of attracting additional activated NK cells. We first demonstrate that the engagement of different activating NK cell receptors, including those mainly involved in tumor cell recognition can induce the active release of HMGB1. Then we show that during NK-mediated tumor cell killing two HMGB1 forms are released, each displaying a specific electrophoretic mobility possibly corresponding to a different redox status. By the comparison of normal and perforin-defective NK cells (which are unable to kill target cells) we demonstrate that, in NK/melanoma cell co-cultures, NK cells specifically release an HMGB1 form that acts as chemoattractant, while dying tumor cells passively release a non-chemotactic HMGB1. Finally, we show that Receptor for Advanced Glycation End products is expressed by NK cells and mediates HMGB1-induced NK cell chemotaxis. Proteomic analysis of NK cells exposed to recombinant HMGB1 revealed that this molecule, besides inducing immediate chemotaxis, also promotes changes in the expression of proteins involved in the regulation of the cytoskeletal network. Importantly, these modifications could be associated with an increased motility of NK cells. Thus, our findings allow the definition of a previously unidentified mechanism used by NK cells to amplify their response to tumors, and provide additional clues for the emerging role of HMGB1 in immunomodulation and tumor immunity.

Human NK cell response to pathogens.

NK cells represent important effectors of the innate immunity in the protection of an individual from microbes. During an NK-mediated anti-microbial response, the final fate (survival or death) of a potential infected target cell depends primarily on the type and the number of receptor/ligand interactions occurring at the effector/target immune synapse. The identification of an array of receptors involved in NK cell triggering has been crucial for a better understanding of the NK cell biology. In this context, NCR play a predominant role in NK cell activation during the process of natural cytotoxicity. Regarding the NK-mediated pathogen recognition and NK cell activation, an emerging concept is represented by the involvement of TLRs and activating KIRs. NK cells express certain TLRs in common with other innate cell types. This would mean that specific TLR ligands are able to promote the simultaneous and synergistic stimulation of these innate cells, providing a coordinated mechanism for regulating the initiation and amplification of immune responses. Evidences have been accumulated indicating that viral infections may have a significant impact on NK cell maturation, promoting the expansion of phenotypically and functionally aberrant NK cell subpopulations. For example, during chronic HIV-infection, an abnormal expansion of a dysfunctional CD56neg NK cell subset has been detected that may explain, at least in part, the defective NK cell-mediated antiviral activity. An analogous imbalance of NK cell subsets has been detected in patients receiving HSCT to cure high risk leukemias and experiencing HCMV infection/reactivation. Remarkably, NK cells developing after CMV reactivation may contain "memory-like" or "long-lived" NK cells that could exert a potent anti-leukemia effect.

TLR/NCR/KIR: Which One to Use and When?

By means of a complex receptor array, Natural killer (NK) cells can recognize variable patterns of ligands and regulate or amplify accordingly their effector functions. Such NK receptors include old, rather conserved, molecules, such as toll-like receptors (TLRs), which enable NK cells to respond both to viral and bacterial products, and newer and evolving molecules, such as killer Ig-like receptors and natural cytotoxicity receptors, which control NK cytotoxicity and are responsible for the elimination of virus-infected or tumor cells without damaging self-unaltered cells. In addition, to rapidly gain new functions NK cells also can acquire new receptors by trogocytosis. Thus, NK cells may have adapted their receptors to different functional needs making them able to play a key role in the modulation of critical events occurring in several compartments of human body (primarily in SLCs but also in decidua during pregnancy). In this review, we will discuss on how the various types of receptors can be used to address specific functions in different immunological contexts.

KIR2DS1-dependent acquisition of CCR7 and migratory properties by human NK cells interacting with allogeneic HLA-C2+ DCs or T-cell blasts.

Natural killer (NK) cells may capture the CCR7 chemokine receptor from allogeneic CCR7(+) cells by trogocytosis and acquire migrating properties in response to lymph node chemokines. This event is negatively regulated by inhibitory killer Ig-like receptors (KIRs) and NKG2A. In this study, we analyzed the role of the HLA-C2-specific activating receptor KIR2DS1 in the process of CCR7 uptake by NK cells interacting with different allogeneic CCR7(+) cells. Co-incubation of KIR2DS1(+) fresh NK cells or NK-cell clones with HLA-C2(+) CCR7(+) lymphoblastoid cell lines resulted in increased CCR7 uptake. Remarkably, KIR2DS1 expression represented a major advantage for acquiring CCR7 from HLA-C2(+) allogeneic dendritic cells (DCs) and T-cell blasts. These findings have important implications in haploidentical hematopoietic stem cell transplantation in which donor-derived (alloreactive) KIR2DS1(+) NK cells, upon CCR7 acquisition, become capable of migrating toward lymph nodes, where they may kill patient DCs and T cells, preventing graft-versus-host and host-versus-graft reactions.

NK/DC crosstalk in anti-viral response.

In recent years, it has been emphasized the role of the crosstalk between natural killer (NK) cells and monocyte-derived dendritic cells dendritic cells (moDCs) in the regulation of the early phases of innate immunity innate immunity and of the subsequent adaptive immune responses. NK cells and DCs coordinate their response communicating through direct cell-to-cell contact and soluble factors. NK cells appear to contribute to the quality control of immature DCs (iDCs) undergoing maturation. On the other hand, DCs may shape the magnitude of innate immune responses by modulating the NK-mediated cytolytic activity against tumors or infected cells. Recent studies suggest that the cooperation between NK cells and DCs is also critical in several anti-viral responses. In particular, NK cells are capable of effectively counteracting viral immune evasion immune evasion strategies by eliminating infected DCs, that display impaired antigen presenting functions, thus indirectly favoring the development of adaptive immune responses to viral antigens cross-presented by healthy DCs.

Bridging innate NK cell functions with adaptive immunity.

Killer Ig-like receptors (KIRs) are major human NK receptors displaying either inhibitory or activating functions which recognize allotypic determinants of HLA-class I molecules. Surprisingly, NK cell treatment with CpG-ODN (TLR9 ligands) results in selective down-modulation of KIR3DL2, its co-internalization with CpG-ODN and its translocation to TLR9-rich early endosomes. This novel KIR-associated function may offer clues to better understand the possible role of certain KIRs and also emphasizes the involvement of NK cells in the course of microbial infections. NK cells are involved not only in innate immune responses against viruses and tumors but also participate in the complex network of cell-to cell interaction that leads to the development of adaptive immune responses. In this context the interaction of NK cells with DC appears to play a crucial role in the acquisition of CCR7, a chemokine receptor that enables NK cells to migrate towards lymph nodes in response to CCL19 and/or CCL21. Analysis of NK cell clones revealed that KIR-mismatched but not KIR-matched NK cells acquire CCR7. These data have important implications in haploidentical haematopoietic stem cell transplantation (HSCT), in which KIR-mismatched NK cells may acquire the ability to migrate to secondary lymphoid compartments (SLCs), where they can kill recipient's antigen presenting cells (APCs) and T cells thus preventing graft versus host (and host vs. graft) reactions.