Subpopulations of NK cells: changes with immunosenescence

La inmunosenescencia se define como un deterioro progresivo de la función inmune asociada a la edad, este estado produce cambios que dan lugar a una remodelación del sistema inmunológico que afecta a la inmunidad innata y adaptativa. Sin embargo, además del envejecimiento “per se” como un factor inductor de estos cambios, las células del sistema inmune sufren un tipo de inmunosenescencia prematura en diversas situaciones de activación crónica del sistema inmune, como infecciones virales, enfermedades autoinmunes y cáncer. En este trabajo analizamos el efecto de la edad, la infección por citomegalovirus (CMV) y el impacto de la leucemia mieloide crónica (LMC) sobre la frecuencia, fenotipo y función de subpoblaciones de células NK. Nuestros resultados confirman la disminución de la subpoblación de células NK CD56bright asociada al envejecimiento y el incremento de la subpoblación CD56-CD16+, así como un incremento de la expresión de CD57 en las subpoblaciones de células NK CD56dimCD16+ y CD56-CD16+. En individuos jóvenes, la seropositividad a CMV no afecta la distribución de las subpoblaciones NK CD56bright, CD56dimCD16+ y CD56-CD16+, mientras que se asocia a un incremento de la expresión de CD57 en las subpoblaciones de células NK CD56dimCD16+ y CD56-CD16+. El estudio de otros receptores NK en estas subpoblaciones celulares muestra una menor expresión de DNAM-1 en células NK CD56dim relacionada con el envejecimiento en individuos CMV seropositivos. Asimismo, la comparación de la expresión de receptores, en subpoblaciones NK de individuos jóvenes según la infección por CMV, muestra un aumento de NKp46 en la subpoblación NK CD56bright y una disminución de la expresión de NKp30 en células NK CD56dim. El descenso de la expresión de NKp30 asociado a la infección por CMV y el descenso de la expresión de DNAM-1 asociado a la edad se observan especialmente en las subpoblaciones de células que coexpresan CD57+. La expresión de NKp46 y NKp30 es menor en células NK CD56dimCD57+ que en las células CD56dimCD57-, mientras que la expresión de DNAM- 1 es mayor en células NK CD57+. La activación in vitro de células NK por IL-2 aumenta la expresión de NKp46 y NKp30 tanto en la subpoblación CD56dimCD57+ como en la CD56dimCD57-. Por otro lado, hemos estudiado la expresión de los receptores inhibidores CD300a y CD161 en células NK en relación con la edad y el seroestatus CMV. El receptor inhibidor CD300a es expresado por la mayoría de las células NK, si bien las células NK CD56bright expresan niveles más altos de CD300a que las células NK CD56dim. La edad se asocia con un aumento en la expresión de CD300a. La expresión de CD161 en células NK CD56dim se encuentra disminuida en jóvenes CMV seropositivos en comparación con jóvenes CMV seronegativos. En las células NK CD56dim, la seropositividad al CMV se asocia a un mayor porcentaje de células CD57+CD300a+ y una reducción en el porcentaje de células CD161+CD300a+. El estudio de los factores de transcripción T-bet y Eomes en células NK en relación con la edad y la seropositividad al CMV, muestra una disminución de T-bethi en células NK CD56dimCD57+ de individuos jóvenes CMV seropositivos, mientras que la expresión de Eomes aumentó con la seropositividad al CMV en CD56bright de individuos de mediana edad y en CD56dimCD57+/-de individuos jóvenes. La expresión de Eomes disminuyó con el envejecimiento en todos los subconjuntos de células NK de los tres grupos de edad. Por último, hemos analizado la expresión de receptores y marcadores de activación y de diferenciación en las subpoblaciones de células NK CD56bright y CD56dim en pacientes con leucemia mieloide crónica (LMC) tratados con inhibidores de tirosina kinasas (tyrosine kinase inhibitors, TKI). Mientras que encontramos diferencias significativas en el fenotipo y la función de las células NK entre individuos sanos de mediana edad y ancianos, las células NK de pacientes con LMC tratados con TKI no muestran diferencias significativas relacionadas con la edad en la mayoría de los parámetros estudiados, lo que indica que la edad no es una limitación de la recuperación de células NK después del tratamiento con TKI. Nuestros resultados también revelan diferencias en la expresión de receptores NK, marcadores de activación y ensayos funcionales (expresión de CD107a e IFN-γ en células NK estimuladas con la línea celular K562) en células NK de pacientes con LMC tratados con TKI en comparación con controles sanos de la misma edad. En conclusión, tomados en conjunto, estos resultados, indican que la edad y la infección por CMV inducen cambios significativos en la expresión de receptores NK, sugerimos varios enfoques como la determinación del CMV o terapias inmunomoduladoras que pueden facilitar el estudio y manejo clínico, especialmente en individuos de edad avanzada. Además, hemos identificado que en pacientes con LMC tratados con TKI, la edad no es una limitación para la recuperación de los receptores de células NK, lo que permite considerar la posibilidad de potenciar la actividad citotóxica de estas células en futuros tratamientos, especialmente en pacientes de mayor edad, para conseguir una remisión más efectiva de la enfermedad después del cese del tratamiento con TKI.Immunosenescence is defined as a progressive deterioration of the immune function associated with age, this state produces changes that result in a remodeling of the immune system that affects the innate and adaptive immunity. However, in addition to aging "per se" as a factor that induces these changes, the cells of the immune system suffer a type of premature immunosenescence in various situations of chronic activation of the immune system, such as viral infections, autoimmune diseases and cancer. In this paper we analyze the effect of age, cytomegalovirus (CMV) infection and the impact of chronic myeloid leukemia (CML) on the frequency, phenotype and function of NK cell subpopulations. Our results confirm the decrease of the subpopulation of CD56bright NK cells associated with aging and the increase of the CD56-CD16+ subpopulation, as well as an increase in the expression of CD57 in the subpopulations of NK CD56dimCD16+ and CD56-CD16+ cells. In young individuals, seropositivity to CMV does not affect the distribution of the NK CD56bright, CD56dimCD16+ and CD56-CD16+ subpopulations, whereas it is associated with an increase in CD57 expression in the subpopulations of CD56dimCD16+ and CD56-CD16+ NK cells. The study of other NK receptors in these cellular subpopulations shows a lower expression of DNAM-1 in CD56dim NK cells related to aging in CMV seropositive individuals. Besides, the comparison of activation receptors expression in NK subpopulations of young individuals shows an increase of NKp46 in the NK CD56bright subpopulation and a decrease in the expression of NKp30 in CD56dim NK cells. The decrease in NKp30 expression was associated with CMV infection only and the decreases in age-associated expression of DNAM-1 were observed especially in subpopulations of cells co-expressing CD57. The expression of NKp46 and NKp30 were lower in NK CD56dimCD57+ cells than in CD56dimCD57-cells, while the expression of DNAM-1 is higher in CD57+ NK cells. The in-vitro activation of NK cells by IL-2 increases the expression of NKp46 and NKp30 in both subpopulation CD56dimCD57+ and the CD56dimCD57-. On the other hand, we have studied the expression of inhibitory receptors CD300a and CD161 in NK cells in relation to age and CMV seroestatus. The CD300a is expressed by most NK cells, showing higher levels of expression in CD56bright NK cells than NK CD56dim cells. Age is associated with an increase in CD300a expression. The expression of CD161 in NK CD56dim cells is decreased in young seropositive CMV compared to young seronegative CMV. In NK CD56dim cells, CMV seropositivity is associated with a higher percentage of CD57+CD300a+ cells and a reduction in the percentage of CD161+CD300a+ cells. The study of T-bet and Eomes transcription factors in NK cells in relation to age and seropositivity to CMV showed a decrease in T-bethi in NK CD56dimCD57+ cells of Young seropositive CMV individuals, while the expression of Eomes increased with CMV seropositivity in CD56bright of middle-aged individuals and in CD56dimCD57+/-of Young individuals. The expression of Eomes decreased with aging in all subsets of NK cells of the three age groups. Finally, we have analyzed the expression of receptors and markers of activation and differentiation in the subpopulations of CD56bright NK cells and CD56dim in patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKI). While we found significant differences in the phenotype and function of NK cells between healthy middle-aged and elderly individuals, NK cells from patients with CML treated with TKI do not show significant age-related differences in most of the parameters studied, which indicates that age is not a limitation of NK cell recovery after treatment with TKI. Our results also reveal differences in the expression of NK receptors, activation markers and functional assays (expression of CD107a and IFN-γ in NK cells stimulated with the K562 cell line) in NK cells from patients with CML treated with TKI compared with healthy controls of the same age. In conclusion, taken together, these results indicate that age and CMV infection induce significant changes in the expression of NK receptors. We suggest several approaches such as the determination of CMV or immunomodulatory therapies that can facilitate the study and the clinical management, especially in elderly individuals. In addition, we have identified that in patients with CML treated with TKI, age is not a limitation for the recovery of NK cell receptors, which allows us to consider the possibility of enhancing the cytotoxic activity of these cells in future treatments, especially in older patients, to achieve a more effective remission of the disease after cessation of treatment with TKI

Effect of Cytomegalovirus (CMV) and Ageing on T-Bet and Eomes Expression on T-Cell Subsets

The differential impact of ageing and cytomegalovirus (CMV) latent infection on human T-cell subsets remains to some extent controversial. The purpose of this study was to analyse the expression of the transcription factors T-bet and Eomes and CD57 on CD4+, CD4hiCD8lo and CD8+ T-cell subsets in healthy individuals, stratified by age and CMV serostatus. The percentage of CD4+ T-cells expressing T-bet or Eomes was very low, in particular in CD4+ T-cells from young CMV-seronegative individuals, and were higher in CMV-seropositive older individuals, in both CD57− and CD57+ CD4+ T-cells. The study of the minor peripheral blood double-positive CD4hiCD8lo T-cells showed that the percentage of these T-cells expressing both Eomes and T-bet was higher compared to CD4+ T-cells. The percentage of CD4hiCD8lo T-cells expressing T-bet was also associated with CMV seropositivity and the coexpression of Eomes, T-bet and CD57 on CD4hiCD8lo T-cells was only observed in CMV-seropositive donors, supporting the hypothesis that these cells are mature effector memory cells. The percentage of T-cells expressing Eomes and T-bet was higher in CD8+ T-cells than in CD4+ T-cells. The percentages of CD8+ T-cells expressing Eomes and T-bet increased with age in CMV-seronegative and -seropositive individuals and the percentages of CD57− CD8+ and CD57+ CD8+ T-cells coexpressing both transcription factors were similar in the different groups studied. These results support that CMV chronic infection and/or ageing are associated to the expansion of highly differentiated CD4+, CD4hiCD8lo and CD8+ T-cells that differentially express T-bet and Eomes suggesting that the expression of these transcription factors is essential for the generation and development of an effector-memory and effector T lymphocytes involved in conferring protection against chronic CMV infection

DNAM-1 and the TIGIT/PVRIG/TACTILE Axis: Novel Immune Checkpoints for Natural Killer Cell-Based Cancer Immunotherapy

Natural killer (NK) cells are lymphocytes of the innate immune response characterized by their role in the destruction of tumor cells. Activation of NK cells depend on a fine balance between activating and inhibitory signals mediated by different receptors. In recent years, a family of paired receptors that interact with ligands of the Nectin/Nectin-like (Necl) family has attracted great interest. Two of these ligands, Necl-5 (usually termed CD155 or PVR) and Nectin-2 (CD112), frequently expressed on different types of tumor cells, are recognized by a group of receptors expressed on T and NK cells that exert opposite functions after interacting with their ligands. These receptors include DNAM-1 (CD226), TIGIT, TACTILE (CD96) and the recently described PVRIG. Whereas activation through DNAM-1 after recognition of CD155 or CD112 enhances NK cell-mediated cytotoxicity against a wide range of tumor cells, TIGIT recognition of these ligands exerts an inhibitory effect on NK cells by diminishing IFN-γ production, as well as NK cell-mediated cytotoxicity. PVRIG has also been identified as an inhibitory receptor that recognizes CD112 but not CD155. However, little is known about the role of TACTILE as modulator of immune responses in humans. TACTILE control of tumor growth and metastases has been reported in murine models, and it has been suggested that it negatively regulates the anti-tumor functions mediated by DNAM-1. In NK cells from patients with solid cancer and leukemia, it has been observed a decreased expression of DNAM-1 that may shift the balance in favor to the inhibitory receptors TIGIT or PVRIG, further contributing to the diminished NK cell-mediated cytotoxic capacity observed in these patients. Analysis of DNAM-1, TIGIT, TACTILE and PVRIG on human NK cells from solid cancer or leukemia patients will clarify the role of these receptors in cancer surveillance. Overall, it can be speculated that in cancer patients the TIGIT/PVRIG pathways are upregulated and represent novel targets for checkpoint blockade immunotherapy

Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade

Effect of Age on NK Cell Compartment in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors

Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deep molecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56bright and CD56dim NK cell subsets and the expression of CD107a and IFN-γ in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35–65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation

NKT-Like (CD3+CD56+) Cells in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors.

Therapy with Tyrosine Kinase Inhibitors (TKI) aiming stable deep molecular response is the gold standard to treat Chronic Myeloid Leukemia (CML). NKT-like cells (CD3+CD56+) combine characteristics of T and NK cells. The physiopathological role of these cells remains unknown although the literature refers their association with inflammation, autoimmune diseases, and cancer. Since the information regarding the role of NKT-like cells in CML is rare, we aimed at the characterization of these cells in CML patients treated with TKIs. Peripheral blood NKT-like cells from 48 CML patients and 40 healthy donors were analyzed by multiparametric flow cytometry. Functional tests consisting of co-culture with leukemic target cells (K562 cell line) were used to measure degranulation and cytokine production. Our results revealed that NKT-like cells are decreased in treated CML patients, although they present increased expression of activation markers (CD69 and HLA-DR), increased degranulation (CD107a) and impaired IFN-γ production. Significantly alterations on the expression of tumor recognition (NCRs and NKp80), and immune regulation receptors (LAG-3, TIM-3, and CD137) by NKT-like cells were observed in CML patients. Second generation TKIs increased cell activation (CD69) and decreased expression of NKp44 and NKp80 by NKT-like cells from CML patients when compared to Imatinib. CML patients that achieved deep molecular response (MR4.5) presented downregulation of NKp44 and LAG-3. Further studies are needed to clarify the role of these cells as biomarkers of therapy response and also to evaluate their value for discrimination of better candidates for sustained treatment-free remission after TKI discontinuation

Effect of Age on NK Cell Compartment in Chronic Myeloid Leukemia Patients Treated With Tyrosine Kinase Inhibitors

Las células asesinas naturales (NK) son un componente muy importante de la respuesta inmunológica innata que participa en la lisis de las células infectadas con virus y las células tumorales. El envejecimiento tiene un profundo impacto en la frecuencia, fenotipo y función de las células NK. La Leucemia Mieloide Crónica (LMC) es causada por la formación del gen BCR-ABL que codifica la oncoproteína aberrante tirosina quinasa. El tratamiento con inhibidores de la tirosina cinasa (TKI) induce una respuesta molecular profunda duradera. La respuesta al tratamiento y la expectativa de vida es menor en los pacientes mayores con fase crónica de LMC que en los más jóvenes. En este trabajo analizamos las células NK de pacientes con LMC tratados con TKI y controles sanos estratificados según la edad. Hemos analizado la expresión de los receptores NK, los marcadores de activación, la diferenciación de las células NK en subconjuntos de células NK CD56bright y CD56dim y la expresión de CD107a y IFN-g en las células NK estimuladas con K562. Mientras que se encontraron diferencias significativas en el fenotipo y la función de las células NK entre individuos sanos de mediana edad (35-65 años) y ancianos (mayores de 65 años), las células NK de pacientes con LMC tratada con TKI no muestran diferencias significativas relacionadas con la edad en la mayoría de los parámetros estudiados, lo que indica que la edad no es una limitación para la recuperación de las células NK después del tratamiento con TKI. Nuestros resultados también revelaron diferencias en la expresión de los receptores NK, marcadores de activación y ensayos funcionales en las células NK de pacientes con LMC tratados con TKI, en comparación con los controles sanos emparejados con la edad. Estos resultados resaltan la relevancia de las células NK en los pacientes tratados con TKI y la necesidad de un análisis extensivo del efecto del envejecimiento en el fenotipo y la función de las células NK en estos pacientes para definir nuevas estrategias basadas en las células NK dirigidas a controlar la progresión de la LMC y lograr la remisión de la enfermedad a largo plazo después del cese del TKI.Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deepmolecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56bright and CD56dim NK cell subsets and the expression of CD107a and IFN-g in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35–65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation.• Fondos FEDER. Operational Program Competitiveness Factors (COMPETE 2020) y Foundation for Science and Technology POCI-01-0145-FEDER-007440, para Paulo Rodrigues Santos y Manuel Amaro Matos de Santos Rosa • Investigación de Santander Universidades de Santander 2016-2017. Programa Becas Iberoamérica, para Nelson López Sejas • Ministerio de Sanidad. Ayudas PI13/02691 y PI16/01615, para Rafael Solana Lara y Carmen Campos Fernández • Ministerio de Economía y Competitividad. Proyecto SAF2017-87538-R, para María Raquel Tarazona Lafarga • Junta de Extremadura. Proyecto IB16164 y GR18085, cofinanciado por Fondos FEDER, para María Raquel Tarazona LafargapeerReviewe