NKG2D Ligands in Liquid Biopsy: The Importance of Soluble and Vesicle-Bound Proteins for Immune Modulation

ABSTRACT: The identification of biomarkers allowing diagnostics, prognostics and patient classification is still a chal lenge in oncological research for patient management. Improvements in patient survival achieved with immunotherapies substantiate that biomarker studies rely not only on cellular pathways contributing to the pathology, but also on the immune competence of the patient. If these immune molecules can be studied in a non-invasive manner, the benefit for patients and clinicians is obvious. The immune receptor Natural Killer Group 2 Member D (NKG2D) represents one of the main systems involved in direct recognition of tumor cells by effector lymphocytes (T and Natural Killer cells), and in immune evasion. The biology of NKG2D and its ligands comprises a complex network of cellular pathways leading to the expression of these tumor-associated ligands on the cell surface or to their release either as soluble proteins, or in extracellular vesicles that potently inhibit NKG2D-mediated responses. Increased levels of NKG2D-ligands in patient serum correlate with tumor progression and poor prognosis; however, most studies did not test the biochemical form of these molecules. Here we review the biology of the NKG2D receptor and ligands, their role in cancer and in patient response to immunotherapies, as well as the changes provoked in this system by non-immune cancer therapies. Further, we discuss the use of NKG2D-L in liquid biopsy, including methods to analyse vesicle-associated proteins. We propose that the evaluation in cancer patients of the whole NKG2D system can provide crucial information about patient immune competence and risk of tumor progressio

Antígenos leucocitarios como genes candidatos para mejorar la respuesta inmune en cerdos

Leukocyte antigens (CD) have functions related to immune response and are of interest as classical candidate genes for health. Polymorphisms (e.g. SNPs) in these genes may be associated with variation in the immune response and consequently in disease response. This approach is being taken in search of susceptibility genes for swine disease. In addition, these genes may vary between populations, especially where specific adaptation to pathogens has occurred, and are of potential interest in characterising pig biodiversity.Los antígenos leucocitarios (CD) tienen funciones relacionadas con la respuesta inmune y son de interés con genes candidatos clásicos para la salud. Los Polimorfismos (ej. SNPs) en estos genes pueden estar asociados con variaciones en la respuesta inmune y consecuentemente con la respuesta a la enfermedad. Este ensayo se está desarrollando en la búsqueda de susceptibilidades genéticas a enfermedades porcinas. Adicionalmente, estos genes pueden variar entre poblaciones, especialmente en la que han ocurrido adaptaciones a patógenos, y suponen un interés potencial para la caracterización de la biodiversidad porcina

Characterisation of five candidate genes within the ETEC F4ab/ac candidate region in pigs

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) that express the F4ab and F4ac fimbriae is a major contributor to diarrhoea outbreaks in the pig breeding industry, infecting both newborn and weaned piglets. Some pigs are resistant to this infection, and susceptibility is inherited as a simple dominant Mendelian trait. Indentifying the genetics behind this trait will greatly benefit pig welfare as well as the pig breeding industry by providing an opportunity to select against genetically susceptible animals, thereby reducing the number of diarrhoea outbreaks. The trait has recently been mapped by haplotype sharing to a 2.5 Mb region on pig chromosome 13, a region containing 18 annotated genes. FINDINGS: The coding regions of five candidate genes for susceptibility to ETEC F4ab/ac infection (TFRC, ACK1, MUC20, MUC4 and KIAA0226), all located in the 2.5 Mb region, were investigated for the presence of possible causative mutations. A total of 34 polymorphisms were identified in either coding regions or their flanking introns. The genotyping data for two of those were found to perfectly match the genotypes at the ETEC F4ab/ac locus, a G to C polymorphism in intron 11 of TFRC and a C to T silent polymorphism in exon 22 of KIAA0226. Transcriptional profiles of the five genes were investigated in a porcine tissue panel including various intestinal tissues. All five genes were expressed in intestinal tissues at different levels but none of the genes were found differentially expressed between ETEC F4ab/ac resistant and ETEC F4ab/ac susceptible animals in any of the tested tissues. CONCLUSIONS: None of the identified polymorphisms are obvious causative mutations for ETEC F4ab/ac susceptibility, as they have no impact on the level of the overall mRNA expression nor predicted to influence the composition of the amino acids composition. However, we cannot exclude that the five tested genes are bona fide candidate genes for susceptibility to ETEC F4ab/ac infection since the identified polymorphism might affect the translational apparatus, alternative splice forms may exist and post translational mechanisms might contribute to disease susceptibility

NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and n vivo but fail to eliminate leukemiai nitiating cells

[Introduction]: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. [Methods]: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells’ cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. [Results]: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells. [Discussion]: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.This work was supported by a grant from the Instituto de Salud Carlos III to LF PI21/01049, the II and V awards from UNOENTRECIENMIL Foundation, and a grant from CRIS FOUNDATION to Beat Cancer as part of the projects “Cell therapy based on NKG2D-CAR for pediatric leukemia” and “NKG2D-CAR as treatment for pediatric patients suffering from acute leukemia and juvenile myelomonocytic leukemia”. AF, MI-N, AN-Z and CF have been supported by Personnel research grants from CRIS Foundation to beat cancer. CM has been supported by Personnel PhD student grants from the Instituto de Salud Carlos III (ISCIII), PFIS (FI19/00176). MVG is funded by grant PID2021-123795OB-I00 from the Spanish Ministry of Science and Innovation [Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI) / 10.13039/501100011033 and European Regional Development Fund (ERDF)-A way of making Europe] and belongs to cancer-Hub CSIC. MI lab is funded by grant PID2020-114148RB-I00 from the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033,which was in part granted with FEDER funding (EC)

Intravenous administration of BCG in mice promotes natural killer and T cell-mediated antitumor immunity in the lung

Intravesical administration of Bacillus Calmette-Guérin (BCG) was one of the first FDA-approved immunotherapies and remains a standard treatment for bladder cancer. Previous studies have demonstrated that intravenous (IV) administration of BCG is well-tolerated and effective in preventing tuberculosis infection in animals. Here, we examine IV BCG in several preclinical lung tumor models. Our findings demonstrate that BCG inoculation reduced tumor growth and prolonged mouse survival in models of lung melanoma metastasis and orthotopic lung adenocarcinoma. Moreover, IV BCG treatment was well-tolerated with no apparent signs of acute toxicity. Mechanistically, IV BCG induced tumor-specific CD8+ T cell responses, which were dependent on type 1 conventional dendritic cells, as well as NK cell-mediated immunity. Lastly, we also show that IV BCG has an additive effect on anti-PD-L1 checkpoint inhibitor treatment in mouse lung tumors that are otherwise resistant to anti-PD-L1 as monotherapy. Overall, our study demonstrates the potential of systemic IV BCG administration in the treatment of lung tumors, highlighting its ability to enhance immune responses and augment immune checkpoint blockade efficacy

NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells

Introduction Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. Methods In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA ⁻ ) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells’ cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. Results In vitro , we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo , NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo , they exhibited functional properties of leukemia initiating cells. Discussion The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL

DataSheet_1_NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells.pdf [Dataset]

[Introduction]: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation.[Methods]: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells’ cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality.[Results]: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells.[Discussion]: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.Peer reviewe

Bladder cancer immunological background and biomarkers Bases inmunológicas del cáncer vesical y biomarcadores

Bladder cancer was one of the first to have a successful treatment based on immune system stimulation, recognized by patient survival and tumor recurrence data. In addition, bladder tumors are now known to have high antigenic load and are therefore considered to be susceptible to respond well to new immunotherapies. For these reasons, studying the mechanism of action of bladder cancer immunological-based treatments can provide valuable information both to improve their current use and to under stand why they work in some patients while others do not tolerate this therapy or have tumor progression. In this article, we will focus on the immune response generated by treatment of non-muscle invasive bladder tumors with BCG, as well as the relationship between this knowledge and new immunotherapies. We will first describe the main activities of the immune system, to continue with the treatment of bladder cancer with BCG, its mechanism of action and biomarkers. Finally, we will summarize the observations that led to the useof monoclonal antibody immunotherapy in cancer and will describe some of the new immunotherapies in use to treat bladder cancer patients.El cáncer vesical fue uno de los primeros en tener un tratamiento de éxito basado en la estimulación del sistema inmunitario, apoyado por los datos de supervivencia de los pacientes y recurrencia de los tumores. Además, hoy día se sabe que los tumores de vejiga presentan alta carga antigénica y, por ello, se considera que son susceptibles de responder favorablemente a las nuevas inmunoterapias. Por estos motivos, estudiar el mecanismo de acción de los tratamientos inmunológicos de cáncer de vejiga nos puede aportar información muy valiosa tanto para mejorar su uso actual como para comprender por qué funcionan en unos pacientes mientras que otros no toleran la terapia o tienen progresión tumoral. En este artículo vamos a centrarnos en la respuesta inmunitaria generada por el tratamiento de los tumores devejiga no-músculo invasivos con BCG, así como la relación entre estos conocimientos y las nuevas inmunoterapias. Para ello, en primer lugar describiremos las principales actividades del sistema inmunitario para continuar con los fundamentos del tratamiento del cáncer devejiga con BCG, su mecanismo de acción y biomarcadores. Por último, recordaremos las observaciones que llevaron al uso de la inmunoterapia con anticuerpos monoclonales en cáncer y describiremos algunas de las nuevas inmunoterapias que se están introduciendo para tratar cánceres vesicales