Inducible bronchus-associated lymphoid tissue (iBALT) is a tertiary lymphoid structure that resembles secondary lymphoid organs. iBALT is induced in the lung in response to Ag exposure. In some ...cases, such as infection with
, the formation of iBALT structure is indicative of an effective protective immune response. However, with persistent exposure to Ags during chronic inflammation, allergy, or autoimmune diseases, iBALT may be associated with exacerbation of inflammation. iBALT is characterized by well-organized T and B areas enmeshed with conventional dendritic cells, follicular dendritic cells, and stromal cells, usually located surrounding airways or blood vessels. Several of the molecular signals and cellular contributors that mediate formation of iBALT structures have been recently identified. This review will outline the recent findings associated with the formation and maintenance of iBALT and their contributions toward a protective or pathogenic function in pulmonary disease outcome.
Natural killer (NK) cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with interleukin-12 (IL-12), IL-15, and IL-18, ...exhibit potent antitumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CARs) have been used to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that ML differentiation and CAR engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased interferon γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets compared with nonspecific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic and CAR specific and required immunoreceptor tyrosine-based activation motif signaling. Furthermore, 19-CAR-ML NK cells generated from lymphoma patients exhibited improved responses against their autologous lymphomas. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrants further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell-resistant tumors.
Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific ...engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation.
We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated
and
.
We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30
lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and
expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30
lymphomas
and
.
We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30
hematologic malignancies, warranting clinical trials with these novel combinations.
N-803 is an IL15 receptor superagonist complex, designed to optimize
persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8
T cells. Monoclonal ...antibodies (mAbs) direct Fc receptor-bearing immune cells, including NK cells, to recognize and eliminate cancer targets. The ability of IL15R agonists to enhance tumor-targeting mAbs in patients has not been reported previously.
Relapsed/refractory patients with indolent non-Hodgkin lymphoma were treated with rituximab and intravenous or subcutaneous N-803 on an open-label, dose-escalation phase I study using a 3+3 design (NCT02384954). Primary endpoint was maximum tolerated dose. Immune correlates were performed using multidimensional analysis via mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) which simultaneously measures protein and single-cell RNA expression.
This immunotherapy combination was safe and well tolerated and resulted in durable clinical responses including in rituximab-refractory patients. Subcutaneous N-803 plus rituximab induced sustained proliferation, expansion, and activation of peripheral blood NK cells and CD8 T cells, with increased NK cell and T cells present 8 weeks following last N-803 treatment. CITE-seq revealed a therapy-altered NK cell molecular program, including enhancement of AP-1 transcription factor. Furthermore, the monocyte transcriptional program was remodeled with enhanced MHC expression and antigen-presentation genes.
N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.
Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early ...development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages.
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•Induced Eomes deletion results in a rapid decrease in NK cell numbers•Eomes-deleted stage III NK cells exhibit increased apoptosis•Eomes-deleted stage II and III NK cells exhibit differentiation defects•Induced Eomes deletion compromises NK cytotoxicity and MHCI−/− rejection in vivo
The transcription factor Eomes is important for early natural killer (NK) cell development. Wagner et al. utilize an inducible, type 1 ILC-specific cre model to demonstrate a stage-specific role for Eomes in NK cell survival and homeostasis as well as a persistent requirement for Eomes in promoting NK cell cytotoxicity.
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. Single-cell 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 CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and 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.
Treatment of advanced melanoma is a clinical challenge. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently deficient or dysfunctional in ...patients with melanoma. Thus, new strategies are needed to enhance NK-cell antitumor responses. Cytokine-induced memory-like (ML) differentiation overcomes many barriers in the NK-cell therapeutics field, resulting in potent cytotoxicity and enhanced cytokine production against blood cancer targets. However, the preclinical activity of ML NK against solid tumors remains largely undefined.
Phenotypic and functional alterations of blood and advanced melanoma infiltrating NK cells were evaluated using mass cytometry. ML NK cells from healthy donors (HD) and patients with advanced melanoma were evaluated for their ability to produce IFNγ and kill melanoma targets
and
using a xenograft model.
NK cells in advanced melanoma exhibited a decreased cytotoxic potential compared with blood NK cells. ML NK cells differentiated from HD and patients with advanced melanoma displayed enhanced IFNγ production and cytotoxicity against melanoma targets. This included ML differentiation enhancing melanoma patients' NK-cell responses against autologous targets. The ML NK-cell response against melanoma was partially dependent on the NKG2D- and NKp46-activating receptors. Furthermore, in xenograft NSG mouse models, human ML NK cells demonstrated superior control of melanoma, compared with conventional NK cells.
Blood NK cells from allogeneic HD or patients with advanced melanoma can be differentiated into ML NK cells for use as a novel immunotherapeutic treatment for advanced melanoma, which warrants testing in early-phase clinical trials.
Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, ...expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for
-haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including
variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.
Summary Tuberculosis (TB) is one of the most important infectious diseases around the world. Several studies have focused on the identification of correlates of protection against TB. Most of them ...have concentrated on the study of IFN-γ due to its robust association with protection against TB. However, given the complexity of the immune response elicited after Mtb infection, other cytokines should also be considered. In the present study, we evaluated Th1 and Th17 responses and their association with the protection or development of active disease. Therefore, non infected individuals (nonTBi), latently infected individuals (LTBi) and patients with active TB (ATB) were studied. The evaluation of the number of cytokine producing cells by ELISPOT showed a higher number of IFN-γ-producing cells in ATB patients, but no differences were found regarding the number of IL-17 producing cells among studied groups. The evaluation of IFN-γ, IL-2, TNF-α and IL-17 producing CD4+ and CD8+ T cells at 1 day and 6 days of stimulation with mycobacterial antigens suggests the presence of functional signatures associated with latency or active TB. The results presented herein suggest the possible use of the evaluation of Th1-type cytokines, such as IFN-γ and/or TNF-α, as a correlate of protection against TB; however, these results need to be validated for other groups.
Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for ...mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.
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