Background: Immune therapies are emerging as a critical component of cancer treatment, capable of delivering durable and potentially curative responses. While CAR-T cell therapy has proven effective for hematological malignancies, it faces challenges in treating solid tumors due to tumor antigen heterogeneity, an immunosuppressive tumor microenvironment, and physical barriers hindering CAR-T cell infiltration. NK cells, particularly adaptive NK (aNK) cells, offer a promising alternative due to their ability to recognize and kill tumor cells without prior sensitization and their resistance to immunosuppressive environments. Purpose: The study investigates the role of cytokines, specifically IL-21 and IL-15, in enhancing aNK cell expansion and activation using peripheral blood mononuclear cells (PBMCs) from healthy donors and tumor-infiltrating lymphocytes (TILs) from glioblastoma (GBM) patients. Methods: Buffy coats and GBM TILs were collected from Karolinska Hospital. NK cells were isolated and expanded in vitro with IL-15 and IL-21 cytokines and feeder cells (K562 and K562E). Furthermore, tumor lysate was added in the cultures to boost memory responses in aNK cells. NK cell functionality, cytotoxicity, and phenotyping was assessed using flow cytometry and statistical analysis (t-test and two-way ANOVA) used to validate the results. Further animal model was used to validate the cytotoxicity capacities of these cells against GBM tumors using a zebrafish model. Results: IL-21 drives the expansion of aNK better than IL-15 controls, data shown in PBMCs and TILs derived from GBM patients and IPLA OVCA patients. Additionally, the use of tumor lysate as a booster for restimulation further amplifies the cytotoxic capacity of aNK cells against autologous tumors. The zebrafish model validates this method, by decreasing the tumor size in zebrafish animals after 3 days of injection. Conclusion: The results demonstrate that IL-21 is essential for the specific expansion of aNK cells, enhancing their aggressiveness towards tumor cells. Additionally, tumor lysate significantly increases the cytotoxic efficacy of aNK cells upon restimulation with the same tumor cells. These findings suggest that IL-21 plays a crucial role in the specific expansion and activation of aNK cells, enhancing their aggressiveness towards tumor cells. By optimizing the expansion protocol, this method aims to advance the clinical application of aNK cells in immunotherapies for solid tumors, offering a potential solution to the limitations faced by current CAR-T therapies. ### Competing Interest Statement The authors have declared no competing interest.

Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is highly variable and could be mediated by a cross-protective pre-immunity. We identified 14 cross-reactive peptides between SARS-CoV-2 and influenza A H1N1, H3N2, and human herpesvirus (HHV)-6A/B with potential relevance. The H1N1 peptide NGVEGF was identical to a peptide in the most critical receptor binding motif in SARS-CoV-2 spike protein that interacts with the angiotensin converting enzyme 2 receptor. About 62%-73% of COVID-19-negative blood donors in Stockholm had antibodies to this peptide in the early pre-vaccination phase of the pandemic. Seasonal flu vaccination enhanced neutralizing capacity to SARS-CoV-2 and T cell immunity to this peptide. Mathematical modeling taking the estimated pre-immunity levels to flu into account could fully predict pre-Omicron SARS-CoV-2 outbreaks in Stockholm and India. This cross-immunity provides mechanistic explanations to the epidemiological observation that influenza vaccination protected people against early SARS-CoV-2 infections and implies that flu-mediated cross-protective immunity significantly dampened the first SARS-CoV-2 outbreaks.

Colorectal cancer (CRC) is one of the most common and fatal types of cancer. Inflammation promotes CRC development, however, the underlying etiological factors are unknown. Human cytomegalovirus (HCMV), a virus that induces inflammation and other cancer hallmarks, has been detected in several types of malignancy, including CRC. The present study investigated whether HCMV infection was associated with expression of the pro-inflammatory enzymes 5-lipoxygenase (5-LO) and cyclooxygenase-2 (COX-2) and other molecular, genetic and clinicopathological CRC features. The present study assessed 146 individual paraffin-embedded CRC tissue microarray (TMA) cores already characterized for TP53 and KRAS mutations, microsatellite instability (MSI) status, Ki-67 index and EGFR by immunohistochemistry (IHC). The cores were further analyzed by IHC for the expression of two HCMV proteins (Immediate Early, IE and pp65) and the inflammatory markers 5-LO and COX-2. The CRC cell lines Caco-2 and LS-174T were infected with HCMV strain VR1814, treated with antiviral drug ganciclovir (GCV) and/or anti-inflammatory drug celecoxib (CCX) and analyzed by reverse transcription-quantitative PCR and immunofluorescence for 5-LO, COX-2, IE and pp65 transcripts and proteins. HCMV IE and pp65 proteins were detected in similar to 90% of the CRC cases tested; this was correlated with COX-2, 5-LO and KI-67 expression, but not with EGFR immunostaining, TP53 and KRAS mutations or MSI status. In vitro, HCMV infection upregulated 5-LO and COX-2 transcript and proteins in both Caco-2 and LS-174T cells and enhanced cell proliferation as determined by MTT assay. Treatment with GCV and CCX significantly decreased the transcript levels of COX-2, 5-LO, HCMV IE and pp65 in infected cells. HCMV was widely expressed in CRC and may promote inflammation and serve as a potential new target for CRC therapy.

Background Natural Killer (NK) cells are innate immune cells able to reject hematological malignancies and correlate with better prognosis in solid tumors. However, conventional NK (cNK) cell anti-tumor activity is limited by the immune suppressive tumor microenvironment (TME). The subset of NK cells, adaptive NK (aNK) cells have immunological memory and accumulate in cytomegalovirus (CMV) infected individuals.1 These aNK cells can resist immune suppression of the TME.2 Glioblastoma (GBM) is the most aggressive brain tumor that has a very poor dismal prognosis and immunosuppressive TME.3 Methods We develop an artificial 3D model of GBM TME, using U251 tumor cells, myeloid derived suppressor cells (MDSC), and NK cells, to analyze the tumor infiltration and killing by NK cells that have developed memory against autologous tumor antigens presented by dendritic cells (DCs), using the live imaging analyzer Incucyte. Results Our data suggest that aNK cells recognize GBM antigens and we demonstrate that NK cells can infiltrate and kill tumor cells (figure 1), especially when previously primed by antigen loaded DCs. Furthermore, using a flow cytometry-based analysis called Metflow we have discovered that aNK cells have a different metabolic profile than cNK cells. While cNK cells were dysfunctional in the TME when inhibiting glycolysis, aNK cells were still activated relaying on alternative metabolic pathways, such as the TCA cycle (figure 2). Conclusions Our data suggest that aNK cells have a higher metabolic plasticity and resistance to TME, making them a great target for immunotherapy against GBM tumors. References Sun JC, Beilke JN, Lanier LL. Immune memory redefined: characterizing the longevity of natural killer cells. Immunol Rev 2010;236:83–94. Sarhan D, et al. Adaptive NK Cells with Low TIGIT Expression Are Inherently Resistant to Myeloid-Derived Suppressor Cells. Cancer Res 2016;76:5696–5706. Grech N, Dalli T, Mizzi S, Meilak L, Calleja N, Zrinzo A. Rising Incidence of Glioblastoma Multiforme in a Well-Defined Population. Cureus. 2020 May 19;12(5):e8195. doi: 10.7759/cureus.8195. PMID: 32572354; PMCID: PMC7302718. Ethics Approval All research involving human blood was ethical approved by the university research ethic committee.

IntroductionHuman cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive.MethodsWe employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses.ResultsHere, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs.DiscussionThese findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Susceptibility to SARSCoV2 infections is highly variable, ranging from asymptomatic and mild infections in most, to deadly outcome in few. Here, we present evidence that antibodies induced by currently circulating influenza A H1N1 (flu) strains cross react with the most critical receptor binding motif of the SARSCoV2 spike protein that interacts with the ACE2 receptor. About 58 to 68% of blood donors in Stockholm had detectable antibodies to this cross-reactive peptide, NGVEGF, and seasonal flu vaccination trended to enhance binding of inhibitory antibodies to SARSCoV2. This peptide also activated CD8 T cells in 20% of healthy subjects. Eleven additional CD8 T cell peptides that cross react with flu and SARSCoV2 were identified that potentially protect against SARSCoV2 in 40 to 71% of individuals, depending on their HLA type.

Emerging evidence supports a significant association between human cytomegalovirus (HCMV) and human malignancies, suggesting HCMV as a human oncomodulatory virus. HCMV gene products are found in >90% of breast cancer tumors and seem to be correlated with more aggressive disease. The definitive diagnosis of HCMV relies on identification of virus inclusions and/or viral proteins by different techniques including immunohistochemical staining. In order to reduce biases and improve clinical value of HCMV diagnostics in oncological pathology, automation of the procedure is needed and this was the purpose of this study. Tumor specimens from 115 patients treated for primary breast cancer at Akershus University Hospital in Norway were available for the validation of the staining method in this retrospective study. We demonstrate that our method is highly sensitive and delivers excellent reproducibility for staining of HCMV late antigen (LA), which makes this method useful for future routine diagnostics and scientific applications.