Adoptive cell therapy (ACT) has shown promising results in cancer treatment, however, achieving effective ex vivo expansion of potent, functionally active, and cytotoxic T cells remains challenging. To overcome this, we loaded the engineered cytokine Neoleukin-2/15 (Neo2/15) on our recently established artificial antigen-presenting scaffolds (Ag-scaffolds) to expand antigen-specific T cells. Neo2/15 selectively binds to IL-2Rβ/γ receptors, enhancing CD8 + T cell proliferation while limiting regulatory T cell expansion. Our study assessed the efficacy of Neo2/15-loaded Ag-scaffolds (Ag-Neo2/15 scaffolds) in expanding antigen-specific T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors. We optimized Ag-scaffold configurations by varying the number of Neo2/15 molecules loaded on Ag-scaffolds and evaluated their impact on T-cell expansion and functionality. We showed that Ag-Neo2/15 scaffolds promoted significant T-cell expansion, with a comparable frequency of antigen-specific CD8 + T cells compared to IL-2/IL-21-loaded Ag-scaffolds (Ag-IL2/21 scaffolds). The CD8 + T cells expanded with Ag-Neo2/15 scaffolds exhibited potent TNFα and IFNγ production and expressed high levels of α4β7 integrin, a homing molecule which is important for directing T cells to specific tissues, potentially enhancing their therapeutic potential. T cells expanded with Ag-Neo2/15 scaffolds had superior and durable cytotoxicity against tumor target cells compared to T cells expanded with Ag-IL2/21 scaffolds. These findings were further supported by our single-cell analysis revealing that T cells expanded with Ag-Neo2/15 scaffolds had higher cytotoxic scores and lower dysfunctionality scores compared to T cells expanded with Ag-IL2/21 scaffolds. The single-cell analysis also indicated increased expression of genes linked to cell division and enhanced proliferative capacity in Ag-Neo2/15 expanded T cells. Furthermore, TCR clonality analysis demonstrated that Ag-Neo2/15 scaffolds promoted the expansion of functionally superior T-cell clones. The top clones of CD8 + T cells expanded with Ag-Neo2/15 scaffolds exhibited a favorable phenotype, essential for effective antigen recognition and sustained T-cell mediated cytotoxicity. Our findings suggest that Ag-Neo2/15 scaffolds represent an advancement in ACT by producing high-quality, functional antigen-specific T cells. This method has the potential to improve clinical outcomes in cancer therapy by generating large numbers of highly functional T cells, thereby optimizing the balance between cytotoxicity and proliferation capacity with less exhausted T-cells in expansion protocols.

CD8+ T cells are crucial for antitumor immunity. However, their functionality is often altered in higher-risk myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). To understand their role in disease progression, we conducted a comprehensive immunophenotypic analysis of 104 pretreatment bone marrow (BM) samples using mass and flow cytometry. Our findings revealed an increased frequency of CD57+CXCR3+ subset of CD8+ T cells in patients who did not respond to azacitidine (AZA) therapy. Furthermore, an increased baseline frequency (>29%) of the CD57+CXCR3+CD8+ T-cell subset was correlated with poor overall survival. We performed single-cell RNA sequencing to assess the transcriptional profile of BM CD8+ T cells from treatment-naïve patients. The response to AZA was linked to an enrichment of IFN-mediated pathways, whereas nonresponders exhibited a heightened TGF-β signaling signature. These findings suggest that combining AZA with TGF-β signaling inhibitors targeting CD8+ T cells could be a promising therapeutic strategy for patients with higher-risk MDS and AML.
Immunophenotypic analysis identified a BM CD57+CXCR3+ subset of CD8+ T cells associated with response to AZA in patients with MDS and AML. Single-cell RNA sequencing analysis revealed that IFN signaling is linked to the response to treatment, whereas TGF-β signaling is associated with treatment failure, providing insights into new therapeutic approaches.
©2024 The Authors; Published by the American Association for Cancer Research.

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDX) in immunodeficient NSG mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.

Summary Regulatory T cells (T regs ) are a critical immune component guarding against excessive inflammatory responses. During chronic inflammation, T regs fail to control effector T cell responses. The causes of T reg dysfunction in these diseases are poorly characterized and therapies are aimed at blocking aberrant effector responses rather than rescuing T reg function. Here we utilized single-cell RNA sequencing data from patients suffering from chronic skin and colon inflammation to uncover SAT1 , the gene encoding spermidine/spermine N1-acetyltransferase (SSAT), as a novel marker and driver of skin-specific T reg dysfunction during T H 17-mediated inflammation. T regs expressing SAT1 exhibit a tissue-specific inflammation signature and show a proinflammatory effector-like profile. In CRISPRa on healthy human skin-derived T regs increased expression of SAT1 leads to a loss of suppressive function and a switch to a T H 17-like phenotype. This phenotype is induced by co-receptor expression on keratinocytes exposed to a T H 17 microenvironment. Finally, the potential therapeutic impact of targeting SSAT was demonstrated in a mouse model of skin inflammation by inhibiting SSAT pharmacologically, which rescued T reg number and function in the skin and systemically. Together, these data show that SAT1 expression has severe functional consequences on T regs and provides a novel target to treat chronic inflammatory skin disease.