A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated killing and infiltration of tumor spheroids.

Understanding the anti-tumor activity of immune cells and testing cancer immunotherapies requires conditions that are as life-like as possible. The tumor microenvironment (TME) describes a complex sum of cellular and acellular actors that influence both immune cells and tumor cells as well as their interplay. Yet in development phases of new immunotherapies, the screening of drugs and adoptive cell products benefits from reproducible and controlled conditions. Two-dimensional (2D) cell cultures cannot simultaneously meet these two challenges therefore lacking considerably predictive power owing to their artificial nature. Various 3D tumor models have therefore been implemented to mimic the architecture and intrinsic heterogeneity of a microtumor. This protocol provides an easy-to-follow, time-efficient, material-limited method for live cell killing and infiltration of single tumor spheroids. It uses multicellular tumor spheroids grown scaffold-free and allows co-culture with immune cells. This protocol is optimized for natural killer (NK) cell functionality assays. However, it can be transferred to other immune cells, in particular cytotoxic T cells. This assay can be analysed using life cell imaging (here with the IncuCyte S3 system) and/or flow cytometry.

Tumor MHC class I expression alters cancer-associated myelopoiesis driven by host NK cells.

Downregulation of MHC class I (MHCI) molecules on tumor cells is recognized as a resistance mechanism of cancer immunotherapy. Given that MHCI molecules are potent regulators of immune responses, we postulated that the expression of MHCI by tumor cells influences systemic immune responses. Accordingly, mice-bearing MHCI-deficient tumor cells showed reduced tumor-associated extramedullary myelopoiesis in the spleen. Depletion of natural killer (NK) cells abrogated these differences, suggesting an integral role of immune-regulatory NK cells during tumor progression. Cytokine-profiling revealed an upregulation of TNF-α by NK cells in tumors and spleen in mice-bearing MHCI expressing tumors, and inhibition of TNF-α enhanced host myelopoiesis in mice receiving adoptive transfer of tumor-experienced NK cells. Our study highlights a critical role of NK cells beyond its identity as a killer lymphocyte and more importantly, the potential host responses to a localized tumor as determined by its MHCI expression.

Process Development for Adoptive Cell Therapy in Academia: A Pipeline for Clinical-Scale Manufacturing of Multiple TCR-T Cell Products.

Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8-99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells.

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development.

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

Improved survival in multiple Myeloma patients undergoing autologous stem cell transplantation is entirely in the standard cytogenetic risk groups.

INTRODUCTION: Novel drugs and drug combinations have improved outcomes for multiple myeloma patients. However, subgroups of patients still have a poor progression-free survival (PFS) and overall survival (OS). In an attempt to identify how the novel drugs affect the outcome in standard-risk and high-risk patients, respectively, we have investigated 715 multiple myeloma (MM) patients who have undergone high dose treatment followed by autologous stem cell transplantation at our center during 1995 - 2020. Outcomes during three time periods, 1995-1999 (period I), 2000-2009 (period II), and 2010-2020 (period III), were compared separately for standard-risk and high-risk patients. Risk stratification was based on chromosome analysis for periods II and III. RESULTS: The whole cohort of patients showed significantly improved OS with time during the three periods being at a median of 5.8, 7.0, and 10.0 years, respectively. There is also a weak tendency for improved PFS, that is, a median of 2.4, 2.6, and 2.9 years, respectively, during the same periods. However, the separate analysis of standard-risk and high-risk patients showed that the overall improvement with time was due to improved standard-risk patients (median OS 8.4 years for the period I and not reached for period II and III). In contrast, no significant improvement was seen in high-risk patients. For patients with del17p, PFS was even worse during period III as compared to period II (median 1.6 vs 3.2 years respectively). CONCLUSION: Our results show that the dramatic improvement in outcome for MM patients during the last 20 years only applies for standard-risk patients, while high-risk MM patients still are doing poorly, indicating that the novel drugs developed during this time are preferentially effective in standard-risk patients. New treatment modalities like CAR-T cells, CAR-NK cells, and/or bispecific antibodies should be tried in clinical studies early in the course of the disease, especially in patients with high-risk cytogenetics.

The Effect of Mesenchymal Stromal Cells Derived From Endometriotic Lesions on Natural Killer Cell Function.

Endometriosis is an inflammatory disease that presents with ectopic endometriotic lesions. Reduced immunosurveillance of these lesions has been proposed to be playing a role in the pathology of endometriosis. Mesenchymal stromal cells (MSC) are found in ectopic lesions and may decrease immunosurveillance. In the present study, we examined if MSC contribute to reduced immunosurveillance through their immunosuppressive effects on natural killer (NK) cells. Stromal cells from endometriotic ovarian cysts (ESCcyst) and eutopic endometrium (ESCendo) of women with endometriosis and their conditioned medium were used in co-cultures with allogeneic peripheral blood NK cells. Following culture, NK cells were examined phenotypically for their expression of activating, inhibitory, maturation, and adhesion receptors and co-receptors, as well as the degranulation (CD107a) marker and the immunostimulatory (interferon-γ) and immunosuppressive (transforming growth factor beta 1 and interleukin-10) cytokines. Moreover, NK cell cytotoxicity was examined using chromium 51 release killing assays. There were no differences between ESCcyst and ESCendo regarding their effects on NK cell cytotoxicity in both conditioned medium and direct co-culture experiments. Additionally, there were no differences between ESCcyst and ESCendo regarding their impact on NK cells' phenotype and degranulation in both conditioned medium and direct co-culture experiments. Although there were no differences found for DNAX accessory molecule-1 (DNAM-1) and NKp44, we found that the expression of the NK cell ligand CD155 that binds DNAM-1 and proliferating cell nuclear antigen (PCNA) that binds NKp44 was significantly less on ESCcyst than on ESCendo. These findings were not supported by the results that the expression of the known and unknown ligands on ESCcyst for DNAM-1 and NKp44 using chimeric proteins was not significantly different compared to ESCendo. In conclusion, the results suggest that ectopic MSC may not contribute to reduced immunosurveillance in endometriosis through their inhibitory effects on NK cells. This suggests that NK cell inhibition in the pelvic cavity of women with endometriosis develops due to other factors.

Comparative evaluation of involved free light chain and monoclonal spike as markers for progression from monoclonal gammopathy of undetermined significance to multiple myeloma.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant clonal plasma cell disorder, with a 1% yearly risk of progression to multiple myeloma (MM). Evolution of M-spike and serum free light chain (sFLC) during follow-up could identify patients at high risk of progression. In this region-wide study, including 4756 individuals, 987 patients with MGUS were identified, and baseline factors as well as evolving involved FLC (iFLC) were evaluated as potential markers for risk of progression from MGUS to MM. Furthermore, evolving iFLC and M-spike were assessed quarterly for a median of 5 years. At baseline, patients that progressed had significantly higher iFLC compared to non-progressors. The risk factors of M-spike >1.5 g/dL, age >65 years and iFLC >100 mg/L were all independently associated with increased risk of MGUS to MM progression. For patients that had any two or three risk factors, the 5-year cumulative probability of progression was significantly higher (31%) compared to no risk factors (2%). Evolving iFLC >100 mg/L during follow-up was consistently associated with increased risk of progression. Based on our observations, we propose to include iFLC as a monitoring tool for all MGUS patients. Furthermore, we recommend a quarterly monitoring in all high-risk patients. Finally, we suggest that the risk of MGUS progression should be stratified with age, M-spike, and iFLC at baseline.

Impact of performance status on overall survival in patients with relapsed and/or refractory multiple myeloma: Real-life outcomes of daratumumab treatment.

BACKGROUND: Little is reported on the real-life impact of daratumumab in relapsed and/or refractory multiple myeloma patients (RRMM). We analyzed a cohort of 156 patients who received daratumumab as a single agent concerning ECOG status, eGFR, cytogenetics, lines of prior treatment, and their impact on survival. RESULTS: Eighty-two (53%) patients were triple refractory, 54 (35%) patients were single or double refractory, and 20 (12%) patients were non-refractory. Following daratumumab treatment, the progression-free survival (PFS) in these groups was 7.2%, 11.4%, and 53% (P < .001), and overall survival (OS) was 34%, 73%, and 58% (P < .001) at 36 months, respectively. Poor ECOG, three lines of prior treatment, and triple refractoriness were all associated with inferior PFS and OS in a multivariate analysis including ECOG, high-risk chromosomal aberrations, refractoriness, number of treatment lines, and eGFR. CONCLUSION: Daratumumab remains an attractive treatment option, especially in patients with poor performance and increased frailty. Furthermore, our observations suggest that patients with ECOG 2 and 3 status require additional supportive and/or palliative therapies to compensate for a potentially effective but encompassing late-line therapy. In conclusion, further prospective studies are needed to elucidate the impact of ECOG 2 and 3 status in patients with RRMM.

Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

CD73 immune checkpoint defines regulatory NK cells within the tumor microenvironment.

High levels of ecto-5'-nucleotidase (CD73) have been implicated in immune suppression and tumor progression, and have also been observed in cancer patients who progress on anti-PD-1 immunotherapy. Although regulatory T cells can express CD73 and inhibit T cell responses via the production of adenosine, less is known about CD73 expression in other immune cell populations. We found that tumor-infiltrating NK cells upregulate CD73 expression and the frequency of these CD73-positive NK cells correlated with larger tumor size in breast cancer patients. In addition, the expression of multiple alternative immune checkpoint receptors including LAG-3, VISTA, PD-1, and PD-L1 was significantly higher in CD73-positive NK cells than in CD73-negative NK cells. Mechanistically, NK cells transport CD73 in intracellular vesicles to the cell surface and the extracellular space via actin polymerization-dependent exocytosis upon engagement of 4-1BBL on tumor cells. These CD73-positive NK cells undergo transcriptional reprogramming and upregulate IL-10 production via STAT3 transcriptional activity, suppressing CD4-positive T cell proliferation and IFN-γ production. Taken together, our results support the notion that tumors can hijack NK cells as a means to escape immunity and that CD73 expression defines an inducible population of NK cells with immunoregulatory properties within the tumor microenvironment.

Iron and Ferritin Modulate MHC Class I Expression and NK Cell Recognition.

The ability of pathogens to sequester iron from their host cells and proteins affects their virulence. Moreover, iron is required for various innate host defense mechanisms as well as for acquired immune responses. Therefore, intracellular iron concentration may influence the interplay between pathogens and immune system. Here, we investigated whether changes in iron concentrations and intracellular ferritin heavy chain (FTH) abundance may modulate the expression of Major Histocompatibility Complex molecules (MHC), and susceptibility to Natural Killer (NK) cell cytotoxicity. FTH downregulation, either by shRNA transfection or iron chelation, led to MHC surface reduction in primary cancer cells and macrophages. On the contrary, mouse embryonic fibroblasts (MEFs) from NCOA4 null mice accumulated FTH for ferritinophagy impairment and displayed MHC class I cell surface overexpression. Low iron concentration, but not FTH, interfered with IFN-γ receptor signaling, preventing the increase of MHC-class I molecules on the membrane by obstructing STAT1 phosphorylation and nuclear translocation. Finally, iron depletion and FTH downregulation increased the target susceptibility of both primary cancer cells and macrophages to NK cell recognition. In conclusion, the reduction of iron and FTH may influence the expression of MHC class I molecules leading to NK cells activation.

Infectious complications and NK cell depletion following daratumumab treatment of Multiple Myeloma.

Treatment with Daratumumab (Dara), a monoclonal anti-CD38 antibody of IgG1 subtype, is effective in patients with multiple myeloma (MM). However, Dara also impairs the cellular immunity, which in turn may lead to higher susceptibility to infections. The exact link between immune impairment and infectious complications is unclear. In this study, we report that nine out of 23 patients (39%) with progressive MM had infectious complications after Dara treatment. Five of these patients had viral infections, two developed with bacterial infections and two with both bacterial and viral infections. Two of the viral infections were exogenous, i.e. acute respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), while five consisted of reactivations, i.e. one herpes simplex (HSV), 1 varicella-zoster (VZV) and three cytomegalovirus (CMV). Infections were solely seen in patients with partial response or worse. Assessment of circulating lymphocytes indicated a selective depletion of NK cells and viral reactivation after Dara treatment, however this finding does not exclude the multiple components of viral immune-surveillance that may get disabled during this monoclonal treatment in this patient cohort. These results suggest that the use of antiviral and antibacterial prophylaxis and screening of the patients should be considered.

Independent control of natural killer cell responsiveness and homeostasis at steady-state by CD11c+ dendritic cells.

During infection and inflammation, dendritic cells (DC) provide priming signals for natural killer (NK) cells via mechanisms distinct from their antigen processing and presentation functions. The influence of DC on resting NK cells, i.e. at steady-state, is less well studied. We here demonstrate that as early as 1 day after DC depletion, NK cells in naïve mice downregulated the NKG2D receptor and showed decreased constitutive phosphorylation of AKT and mTOR. Subsequently, apoptotic NK cells appeared in the spleen concomitant with reduced NK cell numbers. At 4 days after the onset of DC depletion, increased NK cell proliferation was seen in the spleen resulting in an accumulation of Ly49 receptor-negative NK cells. In parallel, NK cell responsiveness to ITAM-mediated triggering and cytokine stimulation dropped across maturation stages, suggestive of a functional deficiency independent from the homeostatic effect. A role for IL-15 in maintaining NK cell function was supported by a gene signature analysis of NK cell from DC-depleted mice as well as by in vivo DC transfer experiments. We propose that DC, by means of IL-15 transpresentation, are required to maintain not only homeostasis, but also function, at steady-state. These processes appear to be regulated independently from each other.

Retuning of Mouse NK Cells after Interference with MHC Class I Sensing Adjusts Self-Tolerance but Preserves Anticancer Response.

Natural killer (NK) cells are most efficient if their targets do not express self MHC class I, because NK cells carry inhibitory receptors that interfere with activating their cytotoxic pathway. Clinicians have taken advantage of this by adoptively transferring haploidentical NK cells into patients to mediate an effective graft-versus-leukemia response. With a similar rationale, antibody blockade of MHC class I-specific inhibitory NK cell receptors is currently being tested in clinical trials. Both approaches are challenged by the emerging concept that NK cells may constantly adapt or "tune" their responsiveness according to the amount of self MHC class I that they sense on surrounding cells. Hence, these therapeutic attempts would initially result in increased killing of tumor cells, but a parallel adaptation process might ultimately lead to impaired antitumor efficacy. We have investigated this question in two mouse models: inhibitory receptor blockade in vivo and adoptive transfer to MHC class I-disparate hosts. We show that changed self-perception via inhibitory receptors in mature NK cells reprograms the reactivity such that tolerance to healthy cells is always preserved. However, reactivity against cancer cells lacking critical MHC class I molecules (missing self-reactivity) still remains or may even be increased. This dissociation between activity against healthy cells and tumor cells may provide an answer as to why NK cells mediate graft-versus-leukemia effects without causing graft-versus-host disease and may also be utilized to improve immunotherapy.

Dynamic Regulation of NK Cell Responsiveness.

Natural killer (NK) cells deliver cytotoxic granules and immunomodulatory cytokines in response to transformed and virally infected cells. NK cell functions are under the control of a large number of germline-encoded receptors that recognize various ligands on target cells, but NK cells also respond to cytokines in the surrounding environment. The interaction between NK cell receptors and their ligands delivers either inhibitory or activating signals. The cytokine milieu further shapes NK cell responses, either directly or by influencing the way inhibitory or activating signals are perceived by NK cells. In this review, we discuss how NK cell function is controlled by inhibitory receptors and MHC-I molecules, how activating receptors contribute to NK cell education, and finally, how cytokines secreted by the surrounding cells affect NK cell responsiveness. Inputs at these three levels involve different cell types and are seamlessly integrated to form a functional NK cell population.