PD-1 is expressed in cytotoxic granules of NK cells and rapidly mobilized to the cell membrane following recognition of tumor cells

5 figures.-- Supplementary information available.The contribution of the T cell-related inhibitory checkpoint PD-1 to the regulation of NK cell activity is still not clear with contradictory results concerning its expression and role in the modulation of NK cell cytotoxicity. We provide novel key findings on the mechanism involved in the regulation of PD-1 expression on NK cell membrane and its functional consequences for the elimination of cancer cells. In contrast to freshly isolated NK cells from cancer patients, those from healthy donors did not express PD-1 on the cell membrane. However, when healthy NK cells were incubated with tumor target cells, membrane PD-1 expression increased, concurrent with the CD107a surface mobilization. This finding suggested that PD-1 was translocated to the cell membrane during NK cell degranulation after contact with target cells. Indeed, cytosolic PD-1 was expressed in freshly-isolated-NK cells and partly co-localized with CD107a and GzmB, confirming that membrane PD-1 corresponded to a pool of preformed PD-1. Moreover, NK cells that had mobilized PD-1 to the cell membrane presented a significantly reduced antitumor activity on PD-L1-expressing-tumor cells in vitro and in vivo, which was partly reversed by using anti-PD-1 blocking antibodies. Our results indicate that NK cells from healthy individuals express cytotoxic granule-associated PD-1, which is rapidly mobilized to the cell membrane after interaction with tumor target cells. This novel finding helps to understand how PD-1 expression is regulated on NK cell membrane and the functional consequences of this expression during the elimination of tumor cells, which will help to design more efficient NK cell-based cancer immunotherapies.Work in the JP laboratory is funded by FEDER (Fondo Europeo de Desarrollo Regional), Gobierno de Aragón (Group B29_20R), Ministerio de Ciencia, Innovación y Universidades (MCUN), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente Inocente, ASPANOA, and Carrera de la Mujer de Monzón. Postdoctoral Juan de la Cierva Contract (MA and LS) and Predoctoral Grant from AECC (CP). JP is supported by ARAID Foundation; Fundación Agencia Aragonesa para la investigación y el Desarrollo; Fundación Científica Asociación Española Contra el Cáncer.Peer reviewe

Adoptive NK cell transfer as a treatment in colorectal cancer patients: analyses of tumour cell determinants correlating with efficacy in vitro and in vivo

6 figures.-- Supplementary material available.Background: Colorectal cancer (CRC) is a heterogeneous disease with variable mutational profile and tumour microenvironment composition that influence tumour progression and response to treatment. While chemoresistant and poorly immunogenic CRC remains a challenge, the development of new strategies guided by biomarkers could help stratify and treat patients. Allogeneic NK cell transfer emerges as an alternative against chemoresistant and poorly immunogenic CRC.Methods: NK cell-related immunological markers were analysed by transcriptomics and immunohistochemistry in human CRC samples and correlated with tumour progression and overall survival. The anti-tumour ability of expanded allogeneic NK cells using a protocol combining cytokines and feeder cells was analysed in vitro and in vivo and correlated with CRC mutational status and the expression of ligands for immune checkpoint (IC) receptors regulating NK cell activity.Results: HLA-I downmodulation and NK cell infiltration correlated with better overall survival in patients with a low-stage (II) microsatellite instability-high (MSI-H) CRC, suggesting a role of HLA-I as a prognosis biomarker and a potential benefit of NK cell immunotherapy. Activated allogeneic NK cells were able to eliminate CRC cultures without PD-1 and TIM-3 restriction but were affected by HLA-I expression. In vivo experiments confirmed the efficacy of the therapy against both HLA+ and HLA− CRC cell lines. Concomitant administration of pembrolizumab failed to improve tumour control.Conclusions: Our results reveal an immunological profile of CRC tumours in which immunogenicity (MSI-H) and immune evasion mechanisms (HLA downmodulation) favour NK cell immunosurveillance at early disease stages. Accordingly, we have shown that allogeneic NK cell therapy can target tumours expressing mutations conferring poor prognosis regardless of the expression of T cell-related inhibitory IC ligands. Overall, this study provides a rationale for a new potential basis for CRC stratification and NK cell-based therapy.Work in the JP laboratory is funded by ASPANOA, CIBER (CB 2021; Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación and Union Europea.NextGenerationEU), Fundacion Inocente, Carrera de la Mujer Monzón, FEDER/Gobierno de Aragón (Group B29_17R), and Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017‐83120‐C2‐1‐R and PID2020-113963RB-I00). Predoctoral grants/contracts from Gobierno de Aragon (IU-M and JP) are supported by ARAID Foundation. EG is funded by Ministerio de Ciencia, Innovación y Universidades (MCNU), and Agencia Estatal de Investigación (PID2020-113963RB-I00). MA and LS are funded by Postdoctoral Juan de la Cierva Contract. SR, LC, SH, and IU-M are funded by predoctoral contracts from Aragon Government. PL is funded by FPU predoctoral grants from Ministerio de Ciencia, Innovación e Universidades. Work at the Catalan Institute of Oncology is funded by the entity, the Instituto de Salud Carlos III and Ministerio de Economia y Competitividad, and co-funded by FEDER funds—a way to build Europe (PI20/00767), CIBERESP (grant CB07/02/2005), H2020 grant MoTriColor, and the Agency for Management of University and Research Grants (AGAUR) of the Catalan Government grant 2017SGR723. This work is supported by COST Action CA17118.Peer reviewe

Biostratigrafía y litoestratigrafía del Paleógieno del área de Sierra Espuña (Cordillera Bética oriental, SE de España)

From new biostratigraphic and lithostratigraphic data, a revision is made of the Paleogene of the Malaguide Complex of Sierra Espuña, located in the Interna1 Zone (Internal-Externa1 Zone Boundary) of the Betic Cordillera. The Paleogene deposits are characterized according to formations and integrated into depositional sequences and sedimentary cycles. The biostratigraphic data are based on larger forarninifers, planktonic foraminifers, and calcareous nannoplankton. The results are integrated into a lithostratigraphic synthesis and correlated to a chronostratigraphic chart proposed by Sena-Kiel et al. (1998). The Paleogene of Sierra Espuña is suhdivided into two main sedimentary cycles hounded by regional unconformities: a preorogenic Lower Sedimentary Cycle and a synorogenic Upper Sedimentary Cycle:1) The Malaguide Tertiary Lower Sedimentary Cycle ranges from the Paleocene to the Lower Oligocene, and is suhdivided into four depositional sequences: The Paleocene Depositional Sequence is represented by the Mula Formation and compnses a lower part dated as Danian, a middle part dated as Selandian, and an upper part dated as Thanetian. The Cuisian-early Lutetian Depositional Sequence is represented by the laterally related Espuña and Valdelaparra formations, which have heen dated as SBZ10-SBZ13(early Cuisian-early Lutetian). The Middle Lutetian-Pnabonian Depositional Sequence is represented by the laterally related Malvariche and Cánovas formations, and has been dated as SBZ14 (middle Lutetian) at the base and NP18-NP20 and P 15 (late Bartonian-Pnahonian) at the top. The Lower Oligocene Depositional Sequence is represented by the As Formation. Our results show that some Lower Oligocene species occur together with reworked Eocene foraminifers in this formation. This age is highly important in determining the true timing of the test events of the upper units of the Malaguide Complex.2) The synorogenic, Malaguide Tertiary Upper Sedimentary Cycle ranges from the late Lower Oligocene to the Aquitanian. It is represented by the laterally related Bosque and Río Pliego Forrnations. The base was dated as NP23 and the upper part as NN 1. This cycle is followed by an also synorogenic, but not malaguide cycle: the Early Burdigalian Sedimentary Cycle represented by the El Niño Formation, which can be correlated with the Viñuela Group.