NK cell education and adhesion molecules : implications for immunotherapy

NK cells have a key role in immune surveillance. They detect aberrant expression of stressinduced molecules and MHC class I molecules with an array of activating and inhibitory receptors. Inhibitory receptors are not only important during the effector phase to guarantee tolerance towards self cells, but also provide the means to learn what self is, during a process termed NK cell education. This mechanism ensures that only NK cells expressing inhibitory receptors for self MHC-I become functionally responsive to react to down-regulation of MHC-I in otherwise normal cells. Recent evidence suggests that education is tunable and changes according to the net signaling input an individual NK cell receives. In paper I, we studied such retuning effects of NK cell education in different settings relevant to immunotherapy and asked whether they would impair anti-tumor responses. We used two mouse models of clinically relevant approaches to interfere with inhibitory receptor signaling, antibody mediated checkpoint blockade of inhibitory receptors, and allogeneic adoptive transfer. Despite adaptation of NK cell responsiveness resulting in tolerance to healthy MHC-I- cells, we could still detect efficient anti-tumor responses. To date, there is no known marker that distinguishes educated from hypo-responsive NK cells. In paper II we investigated the relationship between the adhesion molecule and activating receptor DNAM-1 and the education state of mouse NK cells. DNAM-1 expression appeared early in development prior to inhibitory receptors for MHC-I. We observed in several experimental situations that DNAM-1 levels are higher on NK cells that have Ly49 inhibitory receptors for self MHC-I, and thus are considered to be educated. Furthermore, we found an MHC-I independent, tight correlation between DNAM-1 expression and the inhibitory receptor NKG2A. Based on the data, we propose a model for how DNAM-1 levels may influence NK cell functions in different developmental and functional stages. While NK cell education ensures strong tolerance to most normal cells, one exception is the interaction with DCs. Cross-talk between NK cells and DCs can lead to functional maturation of both cell types, but may also result in NK cell-mediated killing of the DC under certain conditions. In paper III, we analyzed the impact of an inflammatory environment on this interaction. We report that non-inflammatory DCs are relatively resistant to NK cell killing, while under inflammatory conditions, NK cells kill DCs via recognition of CD155 and ICAM-1 by DNAM-1 and LFA-1. In paper IV we explored the possibility of NK cell activation by vaccination with alphagalactosyl-ceramide (αGalCer) loaded exosomes directed to stimulate iNKT cells. We observed a preferential proliferation of educated NK cells in response to iNKT cell activation, as opposed to published results in virus models where preferential proliferation of uneducated cells is observed. This results in increased missing self killing of normal cells, and increased killing of both MHC-I+ and MHC-I- tumor cells. Together, these findings contribute to illustrate the role of NK cells and NK cell education in immune surveillance against cancer cells. The thesis discusses these results in light of NK cells and their application in immunotherapy

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

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-15R beta (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