Natural Killer Receptor Expression Research Articles

NKG2C/KLRC2 tumor cell expression enhances immunotherapeutic efficacy against glioblastoma

BackgroundActivating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the...

The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3-/- CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3-/- and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3-/- CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3-/- CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.

Torque Teno Virus Monitoring in Pediatric Hematopoietic Stem Cell Transplantation

Background Immune reconstitution (IR) after allogeneic hematopoietic stem cell transplantation (HSCT) is crucial to avoid infection, relapse, and graft versus host disease (GvHD). Torque Teno Virus (TTV) is a highly prevalent and ubiquitous small DNA virus which constitutes the main component of human virome. In the transplant setting, TTV viral load (VL) seems to correlate with immunodeficiency status, but most studies have focused on solid organ transplant recipients. Aims To study the kinetics of TTV VL in blood (log10 copies/ml), and its presence in nasopharyngeal aspirate (NPA) by qualitative PCR in children undergoing HSCT, and to study their correlation with HSCT outcomes and other markers of IR in the first year after the procedure. Methods Prospective single center study including patients <18 years old who underwent HSCT in 2020-2022, excluding those with lung disease. Blood and NPA samples were collected pre-HSCT and at days 0, +15, +30, +90, +180, +270 and +360 post-HSCT. Immune cell typing including T/B/NK, and NK phenotyping and cytotoxicity (%CD107a+) were performed in blood, and T/NK cells by flow cytometry and respiratory viruses by multiplex PCR were tested in NPA. TTV in blood was analyzed with TTV R GENE® kits (Biomérieux, France), whereas for NPA an in house PCR developed at the National Centre for Microbiology was used. Results We included 30 patients, whose main characteristics are summarized in Table 1. The most frequent indication for HSCT was malignant disease (76.7%). Donor was mainly HLA mismatch (67%). All patients had engraftment (median 10 days, IQR 10-13.5), but one experienced secondary graft failure. Acute GvHD occurred in 73% of patients (63% grades II-IV), and chronic in 37% (10% severe). Almost all patients (93%) presented at least one episode of infection (median 3, IQR 1-6). Endothelial injury complications occurred in 17%. During the first year post-HSCT, 2/21 patients relapsed. Overall survival was 67% (median follow up 601 days, IQR 424.25-880). No significant differences were found in pre HSCT TTV VL regarding patient age, sex, underlying disease, nor CMV status. TTV VL (Graphic 1) decreased after conditioning to minimum at +15 (mean log10 3.149, SD 1.8), increased from +30 being highest at +270 (mean log10 6.778, SD 1.7), and at +360 was still higher than pre-HSCT (mean log10 6.358, SD 1.9). At day +15, each increase in log10 TTV VL increased the risk of dying 39% (p=0.027). At day +30, TTV VL was higher when total lymphoid irradiation (TLI) was used vs serotherapy (p=0.01). No differences were found regarding GvHD development or infections episodes. TTV could be detected in NPA, although its prevalence seems lower than in blood: 14/44 NPA samples resulted positive (32%), most at day +15 (36%); while 50/182 (27.5%) were positive for respiratory virus, the majority at +270 (28%), without identifying any association with TTV positivity. We found several associations between TTV VL and some parameters of IR: there was an inverse correlation with leukocytes, total, and B lymphocytes at +90 (p=0.001, 0.013, and 0.035 respectively), and with leukocytes at +270 (p=0.042). There was also an inverse correlation at +90 with %CD4+CD45RA+ (p=0.043), and direct with %CD4+CD45RO+ (p=0.038). Finally, TTV VL was direclty associated at pre-HSCT with %CD3+HLADR+ (p=0.035). In our series, associations between TTV VL and NK were positive, the stronger with %CD16+ at +270 (p=0.023). Nevertheless, an inverse correlation was found with %CD107a+ at baseline (p=0.04) and at +15 (p=0.013). There was a lack of a consistent pattern of expression of NK receptors along our study. Conclusions 1. TTV is usually detected in blood in children undergoing HSCT, whereas its detection in NPA is less frequent. 2. TTV could be a potential early biomarker of survival after pediatric HSCT. 3. TTV correlates with less NK citotoxicity before and early after HSCT. 4. TTV VL correlates with slower IR at day 90 after HSCT.

Senescent CD8+ T cells acquire NK cell-like innate functions to promote antitumor immunity.

It has been suggested that aging of the immune system (immunosenescence) results in a decline in the acquired immune response, which is associated with an increase in age-related tumorigenesis. T-cell senescence plays a critical role in immunosenescence and is involved in the age-related decline of the immune function, which increases susceptibility to certain cancers. However, it has been shown that CD8+ T cells with the senescent T-cell phenotype acquire an natural killer (NK) cell-like function and are involved in tumor elimination. Therefore, the role of senescent CD8+ T cells in tumor immunity remains to be elucidated. In this study, we investigated the role of senescent CD8+ T cells in tumor immunity. In a murine model of transferred with B16 melanoma, lung metastasis was significantly suppressed in aged mice (age ≥30 weeks) in comparison to young mice (age 6-10 weeks). We evaluated the cytotoxic activity of CD8+ T cells in vitro and found that CD8+ T cells from aged mice activated in vitro exhibited increased cytotoxic activity in comparison to those from young mice. We used Menin-deficient effector T cells as a model for senescent CD8+ T cells and found that cytotoxic activity and the expression of NK receptors were upregulated in Menin-deficient senescent CD8+ T cells. Furthermore, Menin-deficient CD8+ T cells can eliminate tumor cells in an antigen-independent manner. These results suggest that senescent effector CD8+ T cells may contribute to tumor immunity in the elderly by acquiring NK-like innate immune functions, such as antigen-independent cytotoxic activity.

Human CD8+ T-Cell Populations That Express Natural Killer Receptors.

CD8+ T cells are activated by TCRs that recognize specific cognate Ags, while NK-cell activation is regulated by a balance between signals from germline-encoded activating and inhibitory NK receptors. Through these different processes of Ag recognition, CD8+ T cells and NK cells play distinct roles as adaptive and innate immune cells, respectively. However, some human CD8+ T cells have been found to express activating or inhibitory NK receptors. CD8+ T-cell populations expressing NK receptors straddle the innate-adaptive boundary with their innate-like features. Recent breakthrough technical advances in multi-omics analysis have enabled elucidation of the unique immunologic characteristics of these populations. However, studies have not yet fully clarified the heterogeneity and immunological characteristics of each CD8+ T-cell population expressing NK receptors. Here we aimed to review the current knowledge of various CD8+ T-cell populations expressing NK receptors, and to pave the way for delineating the landscape and identifying the various roles of these T-cell populations.

Bone Marrow Microenvironment Changes in Myelodysplastic Neoplasms and Its Relationship with Clonal Hematopoiesis and Disease Progression

Background: Immune dysregulation and somatic gene mutations are known prognostic factors in myelodysplastic neoplasms (MDS). During the evolution of the disease, a decrease in the immune surveillance is observed, with impaired cytotoxicity and a decrease in mature natural killer cells (NK), among other changes. Also, over expression of inflammatory cytokines, plays a role in the immune environment. The interaction between immune populations and the malignant clone is not entirely understood, especially in the bone marrow. The aims of this study were to characterize bone marrow (BM) immune populations, especially NK cells, and its correlation with the malignant clone. Methods: We prospectively collected 70 BM samples. Fifty MDS without RS or 5q deletion, 11 cytopenia of unknown significance (CUS) and 9 healthy donors (HD). The aims were to analyze NK cells, defined as (CD45+CD3-CD19-) and differentiating an immature population (CD56brigthCD16−NKG2A/CD94+KIR+/−) from a mature one (CD56dim CD16+NKG2A/CD94−KIR+). Their different receptors: activators (NKp46, NKp30, NKG2C, NKG2D, NKp44, DNAM) and inhibitors (TIGIT, NKG2A, Irp60, and PD1), as well as clonal cells expression of their ligands (HLA-ABC, MICA-B, CD155, PD-L1). Finally, KIR haplotype was analyzed, by NGS. Other populations included were myeloid-derived suppressor cells (MDSC), differentiating granulocytic (Gr-MDSC: CD11b+CD33+HLA-DR-CD15+CD14-) and monocytic (Mo-MDSC:CD11b+CD33+ HLA-DRlow/CD15+ CD14+). The analysis was performed, by flow cytometry, on BM fresh samples, following a membrane staining protocol with 10-colors/3 lasers. Molecular analysis was performed by NGS using the Oncomine Myeloid Research Assay included 40 genes. Finally, cytokines concentrations were stablished with the commercial kit ProcartaPlexTM Multiplex immunoassay. Results: A total of 70 subjects (median age of 77 years old and 45% female) were included (Figure 1). Although, total NK was similar among the three groups [MDS 0.17x109/L ICUS 0.07x109/L, HD 0.20x109/L, p= 0.1204], we observed differences between NK subpopulations. An increase of mature NK cells CD56dimCD16+ in CUS (76.16%) compared with MDS (11.58%) and HD (13.67%) [p= 0.0005] was observed. Regarding NK activating receptors, DNAM receptor was highly expressed on MDS (96.05%) and HD (90.98%), compared to CUS (67.79%) [p= 0.007], and the expression of KIR2DS4 was decreased in MDS (28.47%) compared to HD (79.4%) [p= 0.005], as well as NKp44 on MDS (0.19%) compared with HD (0.34%) [p= 0.03]. Interestingly, no expression of KIR2DS4 or NKp44 were observed on CUS. Regarding inhibitor receptors, Irp60 was increased on MDS (95.52%) compared to CUS (85.07%) and HD (80.2%) [p= 0.036] (Figure 2). Inhibitory KIR haplotype AA was observed on 13/47 MDS (27.56%). Finally, no differences were observed between Gr-MDSC [MDS 0.51%, ICUS 0.13%, HD 0.33%, p=0.35] or Mo-MDSC [MDS 1.03%, ICUS 0.25%, HD 0.7%, p=0.28] expression, among groups. Eighty-four percent (42/50) of MDS presented mutations at diagnosis, with a median number of 3. Interestingly, 63.6% (7/11) of CUS presented mutations, 2 DMT3A (VAF < 5%), 1 TET2 (VAF 40%), 1 IDH1 and 1 SF3B1 (VAF 33%), among others. Within subjects presenting mutations, we observed differences on the expression of NKG2A, showing an increase on those with ≥3 mutations (37.23%) vs <3 mutations (35.3%) [p=0.02]. Moreover, differences were found on KIR2DL1 [≥3 mutations 6.66% vs <3 mutations 15.61%, p=0.05]. On the 7 clonal cytopenia of unknown significance (CCUS), no differences were found on the expression of NK receptors compared to idiopathic cytopenia of unknown significance (ICUS). Cytokine analysis on BM of 39 MDS, showed increased levels of IL-10 in HR compared to LR [2 pg/ml vs. 1 pg/ml, p=0.04], including 16 (53%) subjects with very high concentrations (> 40 pg/ml). In addition, we analyzed NK cells of 14 MDS subjects who had a second BM sample, one year after diagnosis. We documented an increase in CD244 NK population [15.23% follow-up vs 3.61% diagnosis, p=0.05]. Conclusions: Our study suggests the presence of immunosuppressive profile of NK cells on MDS, with gain of expression of NKG2A and higher expression of IL10 on high risk MDS. Interestingly, 63.6% CUS subjects were CCUS and showed increase cytotoxic profile. The increase of CD244 expression, at one year control, may be related to an exhausted environment leading to immunosuppression and progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

Abstract 3155: "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for solid tumors

Abstract CAR-T cells expressing chimeric antigen receptors (CAR) have shown clinical success for treating hematologic malignancies, but their efficacy in solid tumors has been limited, largely due to tumor heterogeneity and immunosuppressive tumor microenvironment (TME). Natural Killer (NK) cells derived from human induced pluripotent stem cells (hiPSC) hold great potential to become the next-generation cell therapy products because of their established safety profile and their unique biological activities, especially their potential effectiveness for targeting solid tumor. In contrast to T cells, NK cells exert anti-tumor activity without requiring antigen recognition through MHC complex; they are instead activated by calculated signaling through ligation of multiple activating and inhibitory receptors. As various solid tumors highly express activation ligands for NK cells, we reason that enhanced expression of activation receptors on NK cells could improve their killing activity towards solid tumors. Here, we report that “super NK cells” with more than 10 folds higher expression of NK cell receptors such as NKG2D and NCRs derived from engineered hiPSC show improved cytotoxicity against solid tumors compared with non-engineered ones. The “super NK cells” can achieve more than ten rounds of serial killing against both K562 and THP-1 cell lines in vitro. More importantly, the “super NK cells” show improved cytotoxicity to multiple solid tumors in both in vitro system and in vivo xenograft mouse models. In summary, we have engineered hiPSCs which can be differentiated into "super NK cells" as a potential cell therapy product for improved efficacy against solid tumors. Citation Format: Yiyun Wang, Xiangjun He, Linqin Wang, Mi Shao, Yanan Yue, Yangbin Gao, George Church, He Huang, Luhan Yang. "Super NK cells" - natural killer cells derived from engineered hiPSC with enhanced NK receptor expression demonstrate better anti-tumor effects for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3155.

KIR3DL2 contributes to the typing of acute adult T-cell leukemia and is a potential therapeutic target

AbstractAdult T-cell leukemia (ATL) is a lymphoid neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1), which encodes the transcriptional activator Tax, which participates in the immortalization of infected T cells. ATL is classified into 4 subtypes: smoldering, chronic, acute, and lymphoma. We determined whether natural killer receptors (NKRs) were expressed in ATL. NKR expression (KIR2DL1/2DS1, KIR2DL2/2DL3/2DS2, KIR3DL2, NKG2A, NKG2C, and NKp46) was assessed in a discovery cohort of 21 ATL, and KIR3DL2 was then assessed in 71 patients with ATL. KIR3DL2 was the only NKR among those studied frequently expressed by acute-type vs lymphoma- and chronic/smoldering-type ATL (36 of 40, 4 of 16, and 1 of 15, respectively; P = .001), although acute- and lymphoma-type ATL had similar mutation profiles by targeted exome sequencing. The correlation of KIR3DL2 expression with promoter demethylation was determined by microarray-based DNA methylation profiling. To explore the role of HTLV-1, KIR3DL2 and TAX messenger RNA (mRNA) expression levels were assessed by PrimeFlow RNA in primary ATL and in CD4+ T cells infected with HTLV-1 in vitro. TAX mRNA and KIR3DL2 protein expressions were correlated on ATL cells. HTLV-1 infection triggered KIR3DL2 by CD4+ cells but Tax alone did not induce KIR3DL2 expression. Ex vivo, autologous, antibody-dependent cell cytotoxicity using lacutamab, a first-in-class anti-KIR3DL2 humanized antibody, selectively killed KIR3DL2+ primary ATL cells ex vivo. To conclude, KIR3DL2 expression is associated with acute-type ATL. Transcription of KIR3DL2 may be triggered by HTLV-1 infection and correlates with hypomethylation of the promoter. The benefit of targeting KIR3DL2 with lacutamab is being further explored in a randomized phase 2 study in peripheral T-cell lymphoma, including ATL (registered on https://clinicaltrials.gov as #NCT04984837).

Functionality of natural killer cells in obese asthma phenotypes.

Obesity-associated asthma (OA) is a difficult to treat asthma phenotype due to its severity and poor response to inhaled steroids. Early-onset allergic (EoOA) and late-onset non-allergic (LoOA) OA are suggested subtypes of this phenotype. Natural Killer (NK) cells are key elements of innate immunity involved in cytotoxicity and immune regulation, with uncertain role in OA pathogenesis. Early-onset allergic and LoOA patients together with obese non-asthmatic (ONA) controls have been enrolled in the study. Peripheral blood samples have been collected for analysis. Percentages of total NK cells, CD3- CD56dim and CD3- CD56bright NK cell subsets, cytotoxic activity, intracellular interferon-γ, interleukin (IL)-10, IL-13, IL-17secretion and activatory receptors (NKG2D, NKp46i and NKp44) have been investigated by flow cytometry. The effect of IL-12 and IL-23stimulation on NK cells and intracellular cytokines in different groups have also been analysed and compared with unstimulated conditions. Results of ONA (n=5, age 42±8), EoOA (n=5, age 42±10) and LoOA (n=8, age 46±8) patients have analysed. Body Mass Index has been found to be negatively correlated with CD69(p=.022, r=-0.534). NKG2D receptor has been significantly low in CD56dim cellsof asthma population (p=.046). NKp44 receptor expression has increased after IL-12stimulation in EoOA and control group (p=.02). Intracellular IL-10 content has increased in LoOA and control subjects (p=.018, p=.03) but not in the EoOA group. Intracellular IL-17level has found be higher in allergic OA group. LoOA patients showed a decreased NK cytotoxicity compared with the early-onset asthma group (p=.05). Our study suggests an impaired NK receptor expression, activation and reduced cytotoxicity in OA patients together with variances between different subtypes of this phenotype. This data would be beneficial for tailoring a more personalized treatment strategy combatting steroid resistance and frequent exacerbations in this group of patients.

Investigating the susceptibility of treatment-resistant oesophageal tumours to natural killer cell-mediated responses

The majority of oesophageal adenocarcinoma (OAC) patients do not respond to multimodal treatment regimens and face dismal survival rates. Natural killer (NK) cells are crucial anti-tumour immune cells, and this study investigated the susceptibility of treatment-resistant OAC cells to these potent tumour killers. Natural killer receptor (NKR) ligand expression by OE33CisP (cisplatin-sensitive) and OE33CisR (cisplatin-resistant) cells was investigated. The immunomodulatory effects of OE33CisP and OE33CisR cells on NK cell phenotype and function were assessed. Finally, the impact of chemotherapy regimens on NKR ligand shedding was examined. Our data revealed significantly less surface expression of activating ligands B7-H6, MICA/B, ULBP-3 and activating/inhibitory ligands PVRL-1 and PVRL-4 by OE33CisR cells, compared to OE33CisP cells. Co-culture with OE33CisR cells reduced the frequencies of NKp30+ and NKp46+ NK cells and increased frequencies of TIGIT+, FasL+ and TRAIL+ NK cells. Frequencies of IFN-γ-producing NK cells increased while frequencies of TIM-3+ NK cells decreased after culture with OE33CisP and OE33CisR cells. Frequencies of circulating NKp30+ NK cells were significantly lower in OAC patients with the poorest treatment response and in patients who received FLOT chemotherapy, while B7-H6 shedding by OAC tumour cells was induced by FLOT. Overall, OE33CisR cells express less activating NKR ligands than OE33CisP cells and have differential effects on NKR expression by NK cells. However, neither cell line significantly dampened NK cell cytokine production, death receptor expression or degranulation. In addition, our data indicate that FLOT chemotherapy may promote B7-H6 shedding and immune evasion with detrimental consequences in OAC patients.

Concentration-Dependent Decitabine Effects on Primary NK Cells Viability, Phenotype, and Function in the Absence of Obvious NK Cells Proliferation-Original Article.

Acute myeloid leukemia (AML) cells can evade innate immune killing by modulating natural killer (NK) cells receptors and their cognate ligands in tumor cells, thus it may be possible to restore proper expression of immune receptors or ligands with immune sensitive drugs. Decitabine, as a hypomethylation agent, was approved for the treatment of AML and myelodysplastic syndrome. While clinical responses were contributed by epigenetic effects and the induction of cancer cell apoptosis, decitabine also has immune-mediated anti-tumor effects. After exposure to various concentration of decitabine for 24 h, the primary NK cells (AML-NK cells) cytotoxicity and receptor expression (NKG2D and NKp46) displayed parabola-shaped response, while U-shaped response was seen in cytokine release (IFN-γ and IL-10), and these effects were regulated by ERK and STAT3 phosphorylation level. Furthermore, AML-NK cells function displayed different response when the competitive MEK and STAT3 inhibitors applied respectively. Thus, we could conclude that the different dose of decitabine makes various effects on AML-NK cells function and receptors expression.

Functions of NK and iNKT cells in pediatric and adult CVID, ataxia telangiectasia and agammaglobulinemia patients

Primary immune deficiencies (PID) are known to be more than 400 genetic defects caused by the impairment in development and/or functions of the immune system. Common Variable Immunodeficiency (CVID), Ataxia Telangiectasia (AT) and Agammaglobulinemia (AG) are examples of the most common immunodeficiency syndrome. Natural killer (NK) cells are a component of innate immune system and play a major role in the host-rejection of both tumors and virally infected cells. iNKT cells have a role in autoimmune and infectious diseases and controlling of tumor rejection. In this study, NK and iNKT cells and their functions, and intracellular cytokine amount are aimed to determine in patients that suffer CVID, AT and AG. NKp30, NKp46, NKG2D, perforin and granzyme mRNA expression levels were analyzed using RT-PCR. Receptors, cytokine amount of NK cell subset and iNKT were analyzed by flow cytometry. Decreased CD3+ T and elevated NK cell subset in pediatric AT were found. Expression of NKp44 was decreased in adult AG, but not in pediatric patients. Low NKp44 expression in CD3−CD16+CD56dim NK cell subset was found in pediatric AT patients. High HLA-DR, perforin and granzyme expression were found in CD3−CD16+CD56dim NK cell subset of pediatric CVID and AT patients. Alteration of the number of NK subsets, NK receptor expression and cytokine production were observed in pediatric patients compared to healthy subjects.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Imbalance between Tolerant and Cytotoxic Functional Subsets of NK Cells in a JAK2V617F Myeloproliferative Neoplasm Model

Natural killer (NK) cells are lymphocytes that play a critical role in both innate and adaptive immune responses. It has been shown that CD27 and CD11b are antigens expressed in the last stage of NK cells development and allow the classification of NK cells into three functional subsets. Tolerant NK cells are double negative (DN) for CD27 and CD11b, have an immature phenotype and present low secretory and cytotoxic potential. The secretory NK cells are CD27 positive, have a great ability to release cytokines, but do not exhibit cytotoxicity properties. The cytotoxic NK cells express CD11b, release granzymes and perforins, but are less capable of secreting cytokines. While cytotoxic NK cells are essential for the natural defenses against infected or malignant cells, secretory NK cells, when activated, produce different cytokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10 and others, which may modify the functions or other hematopoietic cells including the hematopoietic stem cells (HSC). We hypothesized that deregulation of the balance between secretory or cytotoxic subtypes of NK cells may favor HSC malignant transformation and contribute to stem cell-derived diseases. In this regard, few studies have shown that NK cells are defective in chronic myeloid leukemia (CML). In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. We aimed to study the activity of NK cells and their subsets in murine primary cells from MPN. NK cells subsets were quantified by flow cytometry by the use of spleen cells from a pre-established conditional vavCre knockin Jak2V617F (Jak2VF) murine model. Besides genotyping to identify controls (Jak2 wt/wt vavCre+, or Jak2WT) and Jak2-mutated mice (Jak2 wt/V617F vavCre+, Jak2VF), the presence of polycythemia and splenomegaly was confirmed in the animals with MPN. Mice (n=5 per group) were euthanized between 8-12 weeks of age. For immunophenotyping, spleen cells were isolated, submitted to red cell lyse, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8, CD3, NK1.1 and CD27 and CD11b. All the stainings were performed at 4°C for 20 minutes and acquisition was performed in a FACSCanto™II flow cytometer (BD Biosciences). Data was analyzed by the use of the Flowjo software version X. When compared to Jak2WT controls, the Jak2VF mice had lower numbers of NK cells (1,447 x 106 versus 0,381 x 106 p<0.05) and T cells (0,638 x 106 versus 0,158 x 106 of the spleen; p<0.05) among the total splenocytes. The analysis of NK subsets showed an increase in the percentages of DN NK cells (CD27-CD11b-) in the spleen of Jak2VF mice as compared to controls (14.84% versus 4.97%; p<0.05). CD27+ immature and mature secretory NK (CD27+CD11b-/+) cells also seemed to be increased in Jak2-mutated mice but this data did not reach statistical significance (37.43% versus 17.65%; p=0.1). On the other hand, when compared to Jak2WT controls, the Jak2VF mice presented lower percentages of CD11b+ cytotoxic NK cells (77.07% versus 47.80% of the spleen cells; p<0.05). In agreement, when the expression of NK receptors was analyzed by flow cytometry, the inhibitory NKG2A receptor was found to be higher expressed in Jak2VF NK cells as compared to non-mutated cells, while the expression of the activatory receptors Ly49H and Ly49D was reduced in Jak2-mutated NK cells. Of note, low levels of the Jak2V617F mutation were detected in sorted NK cells isolated from Jak2VF mice as compared to controls by Real Time quantitative RT-PCR, thus suggesting that the distribution between NK cell subtypes observed here is associated with the JAK2 mutational status. In summary, when compared to controls, Jak2-mutated mice presented an increased proportion of tolerant NK cells, previously described as immature cells with low secretory and cytotolytic potential. To date, CD11b-/CD27- tolerant cells have been observed among solid tumor-infiltrating NK cells, but have not been previously associated with MPN. Moreover, an inverted ratio between the secretory and cytotoxic NK subsets was also detected in Jak2VF animals, suggesting that a cytotoxic deficiency occurs and may contribute to the leukemic stem cell expansion. The expansion of the NK secretory potential deserves further elucidation. Our study provided new insights into the distribution of JAK2V617F NK functional subsets that may be used as future therapeutic targets for MPN. DisclosuresKobayashi:Pfizer: Consultancy.

NKp46 Is a Useful Diagnostic Biomarker in Gastrointestinal T-Cell Lymphoproliferative Diseases and Constitutes a Therapeutic Target. a Celac Network Study

Introduction:Primary gastrointestinal (GI) T-cell lymphoproliferative diseases (T-LPD) are heterogeneous entities, which diagnoses and therapeutic options remain to be standardized. Beside aggressive lymphoma, such as enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), the indolent T-cell lymphoproliferative disease (indolent T-LPD) has been recently described. A differential diagnosis to consider is type II refractory coeliac disease (RCDII) that may complicate CD. Intraepithelial lymphocytes (IEL) from RCDII are dependent for survival on IL-15, which reprograms T-lymphocytes towards a cytotoxic NK phenotype. We previously showed that NKp46 is a new biomarker for both diagnosis and prognosis in GI T-LPD (ICML-2017 Lugano, unpublished).Patients and Methods:The expression of NK receptors was assessed on CD/RCD by flow-cytometry. The number of NKp46 positive IEL per 100 epithelial cells (NKp46+ IEL/100 EC) was studied by immunohistochemistry (IHC) on 195 biopsies from 84 CD/RCD, 44 GI T-cell lymphoma, and on a blinded confirmatory cohort of 48 CD/RCD.8B6A-PBD is an anti-NKp46 monoclonal antibody conjugated (ADC) to a potent cytotoxic drug, pyrrolobenzodiazepine (PBD). For in vivo assessment, tumor-bearing NOD-SCID mice were randomly assigned to PBS, isotype-matched irrelevant Ig-PBD controls, or 8B6A-PBD treatment groups. For ex vivo cytotoxicity assays, isolated cells from fresh small bowel biopsies of RCDII patients were incubated with 8B6A-PBD, Ig-PBD, anti-NKp46 deglycosylated or PBD alone.Results:NKp46 was the only NK receptor that differs between RCDII and CD or RCDI. By doing ROC analysis, a number of NKp46+ IEL/100 EC above 25 clearly discriminated RCDII from CD and RCDI, with excellent positive and negative predictive values of 100 and 95% respectively. Among CD and RCD patients, the survival was shorter if this count is above 25 (OS-5years 72.8% vs. 96.4%, P=0.0004). NKp46 was expressed in EATL (n=20/25) and MEITL (n=4/4) but was never expressed in indolent T-LPD (n=0/15). Positive NKp46 GI T-cell lymphoma had a shorter survival compared to negative NKp46 cases (OS-5years 5.4% vs. 50.5%, P=0.0011).In vivo the anti-NKp46-PBD promoted tumor regression (Fig. B) and prolonged survival (Fig. A) of NOD-SCID mice injected by human NKp46-transfected Raji tumors embedded in matrigel. Ex vivo and as a proof of concept, the anti-NKp46-PBD was highly potent (Fig. D) and selective (Fig. C) against NKp46 positive primary sorted IEL (n=5).Conclusion:NKp46 provides a new biomarker for both diagnosis and prognosis in GI T-LPD. We propose a diagnostic algorithm for GI T-LPD based on the NKp46 expression, which is adapted to immediate, individual patient management. Overall, our results demonstrate for the first time that anti-NKp46 ADC may serve as an effective therapy for RCDII, EATL and MEITL and provide strong preclinical rationale for evaluation of 8B6A-PBD in clinical studies. [Display omitted] DisclosuresCheminant:Innate Pharma: Research Funding. Bruneau:Innate Pharma: Research Funding. Malamut:Cellimmune: Research Funding. Pouyet:MI-mAbs: Employment. Lhospice:Innate Pharma: Employment. Molina:Merck Serono: Honoraria; Takeda: Other: travel support to the ASH meeting; Novartis: Honoraria. Cellier:Cellimmune: Research Funding. Cerf-Bensussan:Cellimmune: Research Funding. Hermine:Novartis: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Hybrigenics: Research Funding; INatherys: Equity Ownership, Research Funding.

Role and Modulation of NK Cells in Multiple Myeloma

Myeloma tumor cells are particularly dependent on their microenvironment and sensitive to cellular antitumor immune response, including natural killer (NK) cells. These later are essential innate lymphocytes implicated in the control of viral infections and cancers. Their cytotoxic activity is regulated by a balance between activating and inhibitory signals resulting from the complex interaction of surface receptors and their respective ligands. Myeloma disease evolution is associated with a progressive alteration of NK cell number, phenotype and cytotoxic functions. We review here the different therapeutic approaches that could restore or enhance NK cell functions in multiple myeloma. First, conventional treatments (immunomodulatory drugs-IMids and proteasome inhibitors) can enhance NK killing of tumor cells by modulating the expression of NK receptors and their corresponding ligands on NK and myeloma cells, respectively. Because of their ability to kill by antibody-dependent cell cytotoxicity, NK cells are important effectors involved in the efficacy of anti-myeloma monoclonal antibodies targeting the tumor antigens CD38, CS1 or BCMA. These complementary mechanisms support the more recent therapeutic combination of IMids or proteasome inhibitors to monoclonal antibodies. We finally discuss the ongoing development of new NK cell-based immunotherapies, such as ex vivo expanded killer cell immunoglobulin-like receptors (KIR)-mismatched NK cells, chimeric antigen receptors (CAR)-NK cells, check point and KIR inhibitors.

Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p&amp;lt;0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p&amp;lt;0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Single Cell Immune Profiling Reveals Distinct T Cell Clones and Functional States in in-Vitro Expanded Cord Blood Derived Gamma Delta T Cells

Gamma-delta (γδ) T cells represent a special class of unconventional T cells defined by their expression of the somatically rearranged T cell receptor (TCR) γ and δ chains. Unlike TCRαβ, it has been reported that different TCRγδ are also able to bind to their antigens in the context of non-classical MHC-like molecules or totally independent of the MHC complexes. Additionally, a variety of NK receptors are known to be expressed by γδ T cells, conferring their ability to sense alternative classes of cancer-associated antigens in a multimodal manner. Such a diverse mode of antigen recognition possibly endows γδ T cells with a wide spectrum of functional activation program. Our team had previously explored the potential of expanding cord blood (CB) derived γδ T cells (CB-gdT) as well as their corresponding ability to target primary acute myeloid leukemia (AML) cells. Using a feeder cell line-based in vitro expansion protocol, we achieved a clinically relevant scale expansion of γδ T cells over a period of 14 days. These cells exhibit variable degree of potency against a range of human AML cell lines and primary patient samples. In order to dissect the cellular and molecular programs governing the activation, differentiation and functional states of our in vitro expanded CB-gdT, we performed multiplex single cell sequencing analysis using the 10X Genomics Chromium System. After initial quality check and filtering, data from a total of 4,276 cells were retrieved. Among which, we identified 742 unique TCRγδ clonotypes, representing 18.6% of the starting 4,000 FACS purified γδ T cells seeded for expansion. The largest 10% of the clones was found to make up 60.9% of the total retrieved cells, demonstrating a significant extent of clonal focusing in our expansion cultures. Consistent to our FACS analysis, Vδ1 is the predominant TRD chain in the expanded cultures, accounting for 61.2% of all clones. Vγ4 is the most prevalent TRG chain making up to 24.9% of all clones regardless of the paired Vδ subtype. Notably, however, the largest γδ T cell clone did not utilize Vγ4, indicating that Vγ4 clones, although frequent, are not the most proliferative clone. These data are supportive of the adaptive characteristics of CB-gdTs, likely in a TCRγδ dependent manner. Based on uniform manifold approximation and projection for dimension reduction (UMAP), all cells were clustered into 11 subsets. Key cytotoxic genes including GZMB, GZMA and NKG7 were all highly expressed across all clusters, indicating that the expanded cells were indeed functionally cytotoxic. Comparing against multiple curated gene sets, we have identified 3 main subsets of γδ T cells: the Proliferative, Cytotoxic γδ T cells (P-CT), Differentiated Cytotoxic γδ T cells (D-CT) and Late Activated Cytotoxic γδ T cells (LA-CT). P-CT (~46% of all cells) shows an expression profile positively associated with cell proliferation as well as increased cell surface expression of memory T cell markers CD27, CCR7 and CD62L. Similar to cytotoxic genes, genes associated with TCR signaling and interferon response were found to be expressed across all cell clusters, yet with elevated levels in D-CT and LA-CT. Furthermore, cell surface expression of different NK receptors including NKG2D, DNAM1 and NKp30 are more enriched in LA-CT compared to the other 2 subsets, suggesting the acquisition of additional NK receptor related functions in this group of cells. Consistent with the concept of progressive γδ T cell differentiation and activation in culture, we found that in 85 (11.5%) of the γδ T cell clones bearing more than 10 cells each, all clones contain cells distributed across the 3 different γδ T cell subsets. Further analysis did not reveal any relationship between the relative proportion of the subsets within each clone with clone size nor any specific type of delta/gamma chain. Taken together, our high-resolution transcriptome analysis suggests that as CB-gdT expand and differentiate in culture, they are likely to adopt dynamic memory and signal -specific functional programs. More importantly, our data highlights the rich clonal and cellular composition of in vitro expanded CB-gdT. These unique characteristics of our CB-gdT can overcome the challenges of tumor heterogeneity and cell persistence, with the potential of improving outcomes in cell immunotherapy. Disclosures Tan: Tessa Therapeutics Ltd: Current Employment.

Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and joint destruction. Previous studies have shown that natural killer (NK) cells may play an important role in the pathogenesis of RA. Interleukin (IL)-15, a pro-inflammatory cytokine which induces proliferation and differentiation of NK cells, is overexpressed in RA. In this present study, we examine various NKRs and adhesion molecule expression on NK cells from RA patients and their response to IL-15 stimulation. We also sought to study cytokine-induced memory-like (CIML) NK cells in RA patients. We established that 1. RA patients had higher NK cell percentages in peripheral blood and their serum IL-15 levels were higher compared to healthy volunteers; 2. NK cells from RA patients showed lower NKp46 expression and an impaired CD69 response to IL-15; 3. NK cells from RA patients showed higher CD158b and CD158e expression but lower CD62L expression; 4. exogenous IL-15 up-regulated CD69, CD158b, CD158e but down-regulated NKp46 and CD62L expression in RA; 5. As to CIML NK cells, restimulation - induced NK cytotoxicity and IFN-γ production was impaired in RA patients, 6. Reduced NKp46, perforin, and granzyme B expression on NK cells was found in RA patients with bone deformity and erosion, 7. RA disease activity (DAS28) showed inverse correlation with the percentages of CD56+CD3− NK cells, and NKp46 and perforin expression on NK cells, respectively. Taken together, our study demonstrated differential expression of various NK receptors in RA patients. NKp46, CD158e, and perforin expression on NK cells may serve as markers of RA severity.