Transplantation of hematopoietic stem cells (HSCT) is a procedure commonly used in treatment of various haematological disorders which is associated with significantly improved survival rates. However, one of its drawbacks is the possibility of development of post-transplant complications, including acute and chronic graft-versus-host disease (GvHD) or CMV infection. Various studies suggested that NK cells and their receptors may affect the transplant outcome. In the present study, patients and donors were found to significantly differ in the distribution of the NKG2A rs7301582 genetic variants - recipients carried the C allele more often than their donors (0.975 vs 0.865, p<0.0001). Increased soluble HLA-E (sHLA-E) levels detected in recipients' serum 30 days after transplantation seemed to play a prognostic and protective role. It was observed that recipients with higher sHLA-E levels were less prone to chronic GvHD (11.65 vs 6.33 pg/mL, p=0.033) or more severe acute GvHD grades II-IV (11.07 vs 8.04 pg/mL, p=0.081). Our results also showed an unfavourable role of HLA-E donor-recipient genetic incompatibility in CMV infection development after transplantation (OR=5.92, p=0.014). Frequencies of NK cells (both CD56dim and CD56bright) expressing NKG2C were elevated in recipients who developed CMV, especially 30 and 90 days post-transplantation (p<0.03). Percentages of NKG2C+ NK cells lacking NKG2A expression were also increased in these patients. Moreover, recipients carrying a NKG2C deletion characterized with decreased frequency of NKG2C+ NK cells (p<0.05). Our study confirms the importance of NK cells in the development of post-transplant complications and highlights the effect of HLA-E and NKG2C genetic variants, sHLA-E serum concentration, as well as NKG2C surface expression on transplant outcome.
Copyright © 2023 Siemaszko, Łacina, Szymczak, Szeremet, Majcherek, Czyż, Sobczyk-Kruszelnicka, Fidyk, Solarska, Nasiłowska-Adamska, Skowrońska, Bieniaszewska, Tomaszewska, Basak, Giebel, Wróbel and Bogunia-Kubik.

Lung cancer remains a devastating disease with a poor clinical outcome. A biomarker signature which could distinguish lung cancer from metastatic disease and detect therapeutic failure would significantly improve patient management and allow for individualized, risk-adjusted therapeutic decisions. In this study, circulating Hsp70 levels were measured using ELISA, and the immunophenotype of the peripheral blood lymphocytes were measured using multiparameter flow cytometry, to identify a predictive biomarker signature for lung cancer patients pre- and post-operatively, in patients with lung metastases and in patients with COPD as an inflammatory lung disease. The lowest Hsp70 concentrations were found in the healthy controls followed by the patients with advanced COPD. Hsp70 levels sequentially increased with an advancing tumor stage and metastatic disease. In the early-recurrence patients, Hsp70 levels started to increase within the first three months after surgery, but remained unaltered in the recurrence-free patients. An early recurrence was associated with a significant drop in B cells and an increase in Tregs, whereas the recurrence-free patients had elevated T and NK cell levels. We conclude that circulating Hsp70 concentrations might have the potential to distinguish lung cancer from metastatic disease, and might be able to predict an advanced tumor stage and early recurrence in lung cancer patients. Further studies with larger patient cohorts and longer follow-up periods are needed to validate Hsp70 and immunophenotypic profiles as predictive biomarker signatures.

Hemogenic endothelium (HE) is the main source of blood cells in the embryo. To improve blood manufacturing from human pluripotent stem cells (hPSCs), it is essential to define the molecular determinants that enhance HE specification and promote development of the desired blood lineage from HE. Here, using SOX18-inducible hPSCs, we revealed that SOX18 forced expression at the mesodermal stage, in contrast to its homolog SOX17, has minimal effects on arterial specification of HE, expression of HOXA genes and lymphoid differentiation. However, forced expression of SOX18 in HE during endothelial-to-hematopoietic transition (EHT) greatly increases NK versus T cell lineage commitment of hematopoietic progenitors (HPs) arising from HE predominantly expanding CD34+CD43+CD235a/CD41a-CD45- multipotent HPs and altering the expression of genes related to T cell and Toll-like receptor signaling. These studies improve our understanding of lymphoid cell specification during EHT and provide a new tool for enhancing NK cell production from hPSCs for immunotherapies.
© 2023 The Author(s).

The role of innate lymphoid cells (ILCs)-including natural killer cells, helper-like ILC1s, ILC2s, ILC3s, and lymphoid tissue inducers-in human cancer is still poorly understood due to the scarcity of cell number. To address this, we present a protocol to analyze or purify ILCs from human blood, adjacent intestine, and colorectal tumor tissue. We describe steps for tissue and blood treatment, density centrifugation, antibody staining, and cell sorting. For complete details on the use and execution of this protocol, please refer to Qi et al. (2021).1.
Copyright © 2023. Published by Elsevier Inc.

Strategies to boost anti-tumor immunity are urgently needed to treat therapy-resistant late-stage cancers, including colorectal cancers (CRCs). Cytokine stimulation and genetic modifications with chimeric antigen receptors (CAR) represent promising strategies to more specifically redirect anti-tumor activities of effector cells like natural killer (NK) and T cells. However, these approaches are critically dependent on tumor-specific antigens while circumventing the suppressive power of the solid tumor microenvironment and avoiding off-tumor toxicities. Previously, we have shown that the stress-inducible heat shock protein 70 (Hsp70) is frequently and specifically expressed on the cell surface of many different, highly aggressive tumors but not normal tissues. We could take advantage of tumors expressing Hsp70 on their membrane ('mHsp70') to attract and engage NK cells after in vitro stimulation with the 14-mer Hsp70 peptide TKDNNLLGRFELSG (TKD) plus low dose interleukin (IL)-2. However, a potential limitation of activated primary NK cells after adoptive transfer is their comparably short life span. T cells are typically long-lived but do not recognize mHsp70 on tumor cells, even after stimulation with TKD/IL-2. To combine the advantages of mHsp70-specificity with longevity, we constructed a CAR having specificity for mHsp70 and retrovirally transduced it into primary T cells. Co-culture of anti-Hsp70 CAR-transduced T cells with mHsp70-positive tumor cells stimulates their functional responsiveness. Herein, we demonstrated that human CRCs with a high mHsp70 expression similarly attract TKD/IL-2 stimulated NK cells and anti-Hsp70 CAR T cells, triggering the release of their lytic effector protein granzyme B (GrB) and the pro-inflammatory cytokine interferon (IFN)-γ, after 4 and 24 hours, respectively. In sum, stimulated NK cells and anti-Hsp70 CAR T cells demonstrated comparable anti-tumor effects, albeit with somewhat differing kinetics. These findings, together with the fact that mHsp70 is expressed on a large variety of different cancer entities, highlight the potential of TKD/IL-2 pre-stimulated NK, as well as anti-Hsp70 CAR T cells to provide a promising direction in the field of targeted, cell-based immunotherapies which can address significant unmet clinical needs in a wide range of cancer settings.
Copyright © 2022 Bashiri Dezfouli, Yazdi, Benmebarek, Schwab, Michaelides, Miccichè, Geerts, Stangl, Klapproth, Wagner, Kobold and Multhoff.