Checkpoint inhibition (CPI) therapy and adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL-based ACT) are the two most effective immunotherapies for the treatment of metastatic melanoma. While CPI has been the dominating therapy in the past decade, TIL-based ACT is beneficial for individuals even after progression on previous immunotherapies. Given that notable differences in response have been made when used as a subsequent treatment, we investigated how the qualities of TILs changed when the ex vivo microenvironment of intact tumor fragments were modulated with checkpoint inhibitors targeting programmed death receptor 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Initially, we show that unmodified TILs from CPI-resistant individuals can be produced, are overwhelmingly terminally differentiated, and are capable of responding to tumor. We then investigate these properties in ex vivo checkpoint modulated TILs finding that that they retain these qualities. Lastly, we confirmed the specificity of the TILs to the highest responding tumor antigens, and identified this reactivity resides largely in CD39+CD69+ terminally differentiated populations. Overall, we found that anti-PD-1 will alter the proliferative capacity while anti-CTLA4 will influence breadth of antigen specificity.
Copyright © 2023 Hulen, Friese, Kristensen, Granhøj, Borch, Peeters, Donia, Andersen, Hadrup, Svane and Met.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs. After two weeks of sequential Wnt and FGF pathway induction, these three-component embryoids are enriched in Pax7-positive embryonic-like myogenic progenitors that can be isolated by flow cytometry. Myogenic differentiation of hiPSCs in heterotypic embryoids relies on a specialized structural microenvironment and depends on MAPK, PI3K/AKT, and Notch signaling. After transplantation in a mouse model of Duchenne muscular dystrophy, embryonic-like myogenic progenitors repopulate the stem cell niche, reactivate after repeated injury, and, compared to adult human myoblasts, display enhanced fusion and lead to increased muscle function. Altogether, we provide a two-week protocol for efficient and scalable suspension-based 3D derivation of Pax7-positive myogenic progenitors from hiPSCs.
© 2022 The Authors.

(1) Background: Prior studies suggested a significant impact of previous live births on peripheral natural killer cells (pNK) in patients with recurrent pregnancy loss (RPL). Patients with primary RPL (pRPL, no live birth) showed higher numbers of pNK than secondary RPL patients (sRPL, ≥ 1 live birth). (2) Methods: To further determine immunological differences between RPL patients and controls, we analysed pNK subpopulations and activation markers in pRPL (n = 47), sRPL (n = 24) and controls with previous live birth (sCtrl, n = 25) and nullipara (pCtrl, n = 60) within a prospective study. Percentages and numbers of CD56dimCD16bright cells, subpopulations and activation markers (CD57+, CD62L+, NKG2D+, NKp46+) were measured in non-pregnant RPL patients and n = 85 controls (n = 60 pCtrl, n = 25 sCtrl) in the mid-luteal phase by flow cytometry. (3) Results: Compared to sRPL patients, sCtrls showed higher CD56+ and CD56dimCD16bright numbers. Further, sRPL patients showed lower numbers of CD56dimCD16brightNKG2D+ and CD56dimCD16brightNKp46+ than sCtrls. (4) Conclusion: We suggest a chronic immune stimulation leading to a lower NK-cell count in sRPL patients with a lower NK cytotoxicity. This underlines the necessity to investigate pNK subpopulations as well as pRPL and sRPL separately to delineate the immune alterations in RPL.

Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94(neg)CD56(dim)NK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive.