Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular ...programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Singlecell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56.sup.bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORCand AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.
Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses and exhibit innate memory following stimulation with IL-12, IL-15, and IL-18, thereby differentiating into ...cytokine-induced memory-like (ML) NK cells. ML NK cells have well-described enhanced anti-tumor properties; however, the molecular mechanisms underlying their enhanced functionality are not well-understood. Initial reports of allogeneic donor ML NK cellular therapy for relapsed/refractory (rel/ref) acute myeloid leukemia (AML) demonstrated safety and a 47% CR/CRi rate (PMID32826231). In this setting, allogeneic ML NK cells are rejected after 3 weeks by recipient T cells, which precludes long-term evaluation of their biology. To address this limitation, we conducted a clinical trial for rel/ref AML patients that added adoptive transfer of same-donor ML NK cells on day +7 of a reduced-intensity conditioning (RIC) MHC-haploidentical HCT, followed by 4 doses of IL-15 (N-803) over 2 weeks (NCT02782546). Since the ML NK cells are from the HCT donor, they are not rejected, but remain MHC-haploidentical to the patient leukemia. Using samples from these patients, we profiled the single cell transcriptomes of NK cells using multidimensional CITE-seq, combining scRNAseq with a custom NK panel of antibodies.
To identify donor ML NK cells in an unbiased fashion, we developed a CITE-seq ML NK classifier from in vitro differentiated paired conventional NK (cNK) and ML NK cells. This classifier was applied via transfer learning to CITE-seq analyzed samples from the donor (cNK cells) and patients at days +28 and +60. This approach identified 28-40% of NK cells as ML at Day +28 post-HCT. Only 1-6% of donor peripheral blood NK cells and 4-7% of NK cells in comparator leukemia patients at day +28 after conventional haplo-HCT alone were identified as ML NK cells (Fig 1A). These ML NK cells had a cell surface receptor profile analogous to a previously reported mass cytometry phenotype. Within the CITE-seq data, ML NK cells expressed a transcriptional profile consistent with enhanced functionality (GZMK, GZMA, GNLY), secreted proteins (LTB, CKLF), a distinct adhesome, and evidence of prior activation (MHC Class II and interferon-inducible genes). ML NK cells had a unique NK receptor repertoire including increased KIR2DL4, KLRC1(NKG2A), CD300A, NCAM1(CD56) , and CD2 with decreased expression of the inhibitory receptor KLRB1(CD161). Furthermore, ML NK cells upregulated HOPX, a transcription factor implicated in memory T cells and murine CMV adaptive NK cells. Additionally, ML NK cells downregulated transcription factors related to terminal maturation (ZEB2) and exhaustion (NR4A2).
We next sought to identify changes during ML differentiation in patients post-HCT from day +28 to +60 post-HCT. Trajectory analysis identified a ML NK cell state distinct from cNK cells that was present at least 60 days post-HCT (Fig 1B). The ML transcriptional phenotype continued to modulate during late differentiation, including downregulation of GZMK and NCAM1, and upregulation of maturation related transcription factors, while maintaining high expression of HOPX. ML NK cells retained their enhanced functionality during in vivo differentiation, as patient ML NK cells had significantly increased IFNγ production compared to cNK cells after restimulation with leukemia targets or cytokines using mass cytometry (Fig. 2). Subsequently, we confirmed the ML CITE-seq profile in an independent clinical trial treating pediatric AML relapsed after allogenic HCT with same-donor ML NK cells (NCT03068819). In this setting, ML NK cells expressed a similar transcriptional signature and persisted for at least 2 months in the absence of exogenous cytokine support. Thus, ML NK cells possess a distinct transcriptional and surface proteomic profile and undergo in vivo differentiation while persisting within patients for at least 2 months. These findings reveal novel and unique aspects of the ML NK cell molecular program, as well as their prolonged functional persistence in vivo in patients, assisting in future clinical trial design.
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Foltz: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Bednarski: Horizon Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fehniger: Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; ImmunityBio: Research Funding; Kiadis: Other; Affimed: Research Funding; Compass Therapeutics: Research Funding; HCW Biologics: Research Funding; OrcaBio: Other; Indapta: Other.
Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of ...tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNgamma produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNgamma levels in HHCs correlate with tuberculosis development. A cohort of 2060 HHCs was followed for 2-3 years after exposure to a tuberculosis case. Besides TST, IFNgamma responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination. Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNgamma response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNgamma responders to CFP-10 (HR 1.82 95% CI 0.79-4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNgamma producers was observed (trend Log rank p = 0.007). CFP-10-induced IFNgamma production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Metastatic (m) colorectal cancer (CRC) is an incurable, frequently lethal disease present in approximately 25% of newly diagnosed CRC patients, and almost 50% of the patients with CRC will ...develop metastatic disease. For mCRC, while systemic therapies (cytotoxic therapy, targeted therapy, immunotherapy or their combinations) have extended patient’s life expectancy, not all patients are candidates for these treatments. Immunotherapy has achieved significant curative effects in patients with solid tumors; however, a considerable proportion of mCRC patients (~90%) are not responsive to immune checkpoint blockade (ICB) leaving a gap in the CRC immunotherapy options.
Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses against a wide variety of tumor cells; however, they are frequently dysfunctional in cancer patients. NK cells from CRC patients exhibit decreased expression of activating receptors and reduced cytokine production after stimulation with CRC cells with a more accentuated phenotype in advanced stages of the disease. Memory-like (ML) NK cells differentiated after IL-12, IL-15, and IL-18 activation have been shown to overcome limitations associated with deficient tumor recognition and poor anti-tumor activity. In clinical trials, ML NK cells were safe and active against acute myeloid leukemia (Romee R et al, Sci Transl Med, 2016). Preclinically, ML NK cells exhibited improved responses against melanoma (Marin ND et al, Clin Cancer Res, 2021) and ovarian cancers, compared to conventional NK cells. Here, we hypothesized that memory-like differentiation will enhance multiple aspects of the NK cell response against CRC cells.
Allogeneic ML NK cells displayed enhanced IFN-γ production against four CRC cell lines DLD-1 (p=0.015), SW480 (p=0.0005), HT-29 (p=0.0005) and HCT116 (p=0.001), as well as primary patient derived CRC tumoroids (p=0.0156), compared to conventional (c) NK cells. ML NK cells also exhibited superior and sustained killing of CRC cell lines over time compared to cNK cells, as measured by Incucyte assays and using CRC tumoroids. Furthermore, IFN-γ production was significantly reduced after blockade of NKG2D, DNAM-1 and NKp46 (p<0.01) revealing mechanistic insights into how ML NK cells recognize CRC targets. Finally, using a xenograft model of CRC in NSG mice, we demonstrated that ML NK cells exhibited superior control of Luciferase expressing HCT116 cells compared to cNK cells (p=0.01) as measured by bioluminescent imaging (BLI).
Collectively, these findings demonstrate that ML NK cells exhibit enhanced responses against CRC cells, and thus warrants further investigation in clinical trials for CRC patients, especially those who are not candidates for standard of care therapy or failed ICB.
Citation Format: Nancy D. Marin, Michelle Becker-Hapak, Quazim A. Alayo, Melissa Berrien-Elliot, Lynne Marsala, Naomi Sonnek, Miriam T. Jacobs, Jennifer A. Foltz, Alice Zhou, Jennifer Tran, Pamela Wong, Celia Cubit, Kimberly Hwang, Timothy Schappe, Ryan C. Fields, Matthew A. Ciorba, Todd A. Fehniger. Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 893.
Abstract
Recent advances in treatment options for multiple myeloma (MM) have improved patient outcomes, however, MM remains incurable and options are limited for relapsed/refractory disease. ...Memory-like (ML) differentiation of NK cells using brief IL-12, IL-15, and IL-18 activation generates a safe allogenic NK cellular therapy for acute myeloid leukemia that induce complete remissions. Here, we investigate pre-clinical combinations with ML NK cells for MM, including blockade of the inhibitory NKG2A/HLA-E checkpoint as well as MM-specific targeting via myeloma-targeting monoclonal antibody (mAb) and chimeric antigen receptor (CAR) engineering.
Human ML NK cells generated from healthy donors display IFN-γ responses similar to conventional (cNK) cells, and only modestly increased cytotoxicity against myeloma cell lines (MM1.s and OPM2). MM cell lines and primary MM cells express high levels of HLA-E, a major negative checkpoint for ML NK cells that binds to the inhibitory receptor NKG2A expressed on NK cells. Indeed, mass cytometry analysis of MM patients compared to healthy controls demonstrated enrichment of a NK cell population expressing high levels of NKG2A, but also high levels of activation and cytotoxic markers. Disruption of HLA-E binding to NKG2A with the blocking mAb monalizumab (AstraZeneca) significantly increased healthy donor ML NK cell IFN-γ response (P=0.016), degranulation (surface CD107a, P=0.017), and killing (P=0.009) of MM1.s when compared to isotype mAb. NK cells isolated from patients with MM showed similarly enhanced functionality after ML differentiation and NKG2A blockade (IFN-γ P=0.008). In an NSG mouse model of MM using MM1.s cells, healthy donor ML NK cells combined with monalizumab showed improved control of MM tumor growth compared to ML NK cells without monalizumab (P<0.001), conventional NK cells, or control tumors alone (P<0.001).
To further improve recognition of MM, ML NK cells responses were assessed in combinationwith a mAb against SLAMF7 (elotuzumab) that both targets MM cells and activates NK cells. Elotuzumab treatment increased the functionality of ML NK cells against MM cell lines (IFN-γ, P=0.004) with an additive effect of both elotuzumab and monalizumab (IFN-γ, P=0.003). In an alternative strategy to enhance MM targeting, we engineered an anti-BCMA CAR (anti-BCMA/4-1bb/z) into ML NK cells. BCMA-CAR+ NK cells exhibited increased IFN-γ (P=0.001) production and degranulation (P<0.001) against MM1.s compared to non-transduced control NK cells. These functional responses were additionally enhanced with ML differentiation.
Collectively, these findings suggest that ML NK cell differentiation, combined with strategies to address inhibitory checkpoints (NKG2A) or MM-specific activation (elotuzumab, anti-BMCA CAR), are promising cellular therapy strategies for MM patients.
Citation Format: Alice Y. Zhou, Melissa M. Berrien-Elliott, Ravi Vij, Mark Fiala, Michelle Becker-Hapak, Lynne Marsala, Miriam T. Jacobs, Nancy D. Marin, Jennifer Tran, Jennifer Foltz, Pamela Wong, Julie Fortier, Sarah Kelley, Carly Neal, David Russler-Germain, Timothy Schappe, Todd A. Fehniger. Overcoming barriers to the natural killer cell response against multiple myeloma: Manipulating the NKG2A/HLA-E checkpoint and CAR engineering of memory-like natural killer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2897.
ABSTRACT Tuberculosis (TB) is one of the leading causes of death due to a single infectious agent. The development of a TB vaccine that induces durable and effective immunity to Mycobacterium ...tuberculosis (Mtb) infection is urgently needed. Early and superior Mtb control can be induced in M. bovis Bacillus Calmette-Guérin (BCG)-vaccinated hosts when the innate immune response is targeted to generate effective vaccine-induced immunity. In the present study, we show that innate activation of DCs is critical for mucosal localization of clonally activated vaccine-induced CD4+ T cells in the lung and superior early Mtb control. In addition, our study reveals that Th1/Th17 cytokine axis play an important role in superior vaccine-induced immunity. Our studies also show that activation of the nuclear factor kappa-light-chain enhancer of activated B cell (NF-κβ) pathway in lung epithelial cells is critical for the mucosal localization of activated vaccine-induced CD4+ T cells for rapid Mtb control. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control. IMPORTANCE Tuberculosis is a leading cause of death due to single infectious agent accounting 1.4 million deaths each year. The only licensed vaccine, BCG, is not effective due to variable efficacy. In our study, we determined the early immune events necessary for achieving complete protection in a BCG-vaccinated host. Our study reveals that innate activation of DCs can mediate superior and early Mtb control in BCG-vaccinated mice through lung epithelial cell signaling and localization of clonal activated, Mtb antigen-specific, cytokine-producing CD4+ T cells within the lung parenchyma and airways. Thus, our study provides novel insights into the immune mechanisms that can overcome TB vaccine bottlenecks and provide early rapid Mtb control.
Abstract
Although CD28 is considered the main T cell costimulatory molecule, CD27 has also costimulatory activity; nevertheless, it is not well established whether they play overlapping or ...complementary roles during CD4 T cell activation. Both molecules are co-expressed on a large percentage of CD4 T cells, mainly in naive and central memory T cells; but their ligands, CD80/CD86 and CD70 respectively, are differentially expressed on antigen presenting cells.
To further differentiate the costimulatory role of CD28 and CD27 in the human CD4 T cell responses, circulating T cells from healthy donors were stimulated with Mycobacterium tuberculosis Purified Protein Derivative (PPD) under conditions of selective blockade of CD80 and CD86 or CD70 binding. The costimulatory activities of CD28 and CD27 in CD4 T cells memory responses were compared by stimulating PBMC, from tuberculin positive (TST+) donors, with PPD in the presence of either anti-CD80 plus anti-CD86 or anti-CD70 and then measuring CD4 T cells proliferation, IFN-gamma production and CD30 expression by flow cytometry. Treatment with anti-CD80 plus anti-86 inhibited all CD4+ T cell responses, but anti-CD70 had no effect. To study their role in the CD4+ T cell primary responses, myeloid dendritic cells, from TST negative donors, were pulsed with PPD and cocultured with autologous T cells under the blockade conditions described above. Anti-CD80 plus anti-CD86 as well as anti-CD70 inhibited CD4+ T cell proliferation and CD30 expression. These results support that CD28-CD80/CD86 signals are needed for both primary and memory CD4 T cell responses, whereas CD27-CD70 signals are required mainly for the primary anti-PPD responses. (Supported by Colciencias, Colombia, contract 0275-2014).
Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-g production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection ...(LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120 h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144 h and the percentage of CD4 super(+) and CD8 super(+)IFN-g super(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4 super(+) and CD8 super(+) precursor cells, as well as decreased number of CD4 super(+)IFN-g super(+) cells in response to all antigens, whereas CD8 super(+)IFN-g super(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-g responses, irrespective of the antigen employed. The CD4 super(+)/CD8 super(+) cell ratios showed that in response to PPD CD4 super(+) precursor and IFN-g-producer cells are more frequent than their CD8 super(+) counterparts, and that PTB have a decreased CD4 super(+)IFN-g super(+)/CD8 super(+)IFN-g super(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4 super(+)IFN-g super(+) and CD8 super(+)IFN-g super(+) cells exhibited a central memory phenotype (CD45RO super(+)CD27 super(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4 super(+) and CD8 super(+) precursor cells and CD4 super(+)IFN-g super(+). HHCs exhibited the highest frequency of CD4 super(+) and CD8 super(+) precursors and CD4 super(+)IFN-g super(+)-producing cells.
Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of ...tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNgamma produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNgamma levels in HHCs correlate with tuberculosis development.
A cohort of 2060 HHCs was followed for 2-3 years after exposure to a tuberculosis case. Besides TST, IFNgamma responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination.
Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNgamma response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNgamma responders to CFP-10 (HR 1.82 95% CI 0.79-4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNgamma producers was observed (trend Log rank p = 0.007).
CFP-10-induced IFNgamma production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprophylaxis to child contacts regardless of BCG vaccination.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of ...tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNg produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNg levels in HHCs correlate with tuberculosis development. A cohort of 2060 HHCs was followed for 2-3 years after exposure to a tuberculosis case. Besides TST, IFNg responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellin, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination. Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p&0.0001). IFNg response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR=6.07, p&0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children &5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNg responders to CFP-10 (HR 1.82 95% CI 0.79-4.20 p=0.16). However, a significant trend for tuberculosis development amongst high HHC IFNg producers was observed (trend Log rank p=0.007). CFP-10-induced IFNg production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprohylaxis to child contacts regardless of BCG vaccination.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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