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.

Isolation of viable immune cells from tissues is critically important to characterize cellular and molecular processes during homeostasis and disease. Here, we provide an optimized protocol to achieve high yields of viable intestinal lamina propria mononuclear cells (LPMCs). We describe steps for intestinal tissue collection from humans and nonhuman primates, followed by mechanical disruption and enzymatic digestion. Furthermore, we detail characterization of the mononuclear phagocyte (MP) subtypes by flow cytometry analysis. The protocol is repeatable and scalable for downstream applications. For complete details on the use and execution of this protocol, please refer to Cavarelli et al. (2022).
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Severe coronavirus disease 2019 (COVID-19) is linked to insufficient control of viral replication and excessive inflammation driven by an unbalanced immune response. Plasmacytoid dendritic cells (pDCs) are specialized in the rapid production of interferons in response to viral infections, and can also prime and activate T-cells. Conventional DCs (cDCs) are critical for the elimination of viral infections owing to their specialized ability to prime and activate T cells. We assessed the frequency and phenotype of pDCs and cDCs in survivors and non-survivors of COVID-19.
Patients with COVID-19 were enrolled, and 22 were included in this study. Peripheral whole blood was obtained during the 2nd week of illness, stained with antibodies specific for lineage markers, human leukocyte antigen-DR isotype (HLA-DR), CD11c, and CD123, and analyzed by flow cytometry. Patients were followed-up during hospital admission and grouped into survivors (n=17) and non-survivors (n=5) of COVID-19.
The ratio of pDCs to pre-cDCs was significantly lower (P=0.0005) in non-survivors compared to survivors. The frequency of pDCs was significantly higher than cDC2-like cells (P=0.0002) and pre-cDCs (P<0.0001) in survivors but not in non-survivors. HLA-DR expression level on pDCs and cDC2-like cells was lower in non-survivors compared to survivors (P=0.02 and P=0.058, respectively), and HLA-DR was inversely correlated with disease severity rating (pDCs: r= -0.47, P=0.027; cDC2-like cells: r= -0.45, P=0.037). CD123 expression level on pDCs was significantly lower (P=0.038) in non-survivors compared to survivors, and CD123 was inversely correlated with disease severity rating (r=-0.5, P=0.016). CD11c expression level on cDC2-like cells was significantly lower (P=0.03) in non-survivors compared to survivors, and CD11c was inversely correlated with disease severity rating (r=-0.47, P=0.025).
A lower frequency of pDCs compared to other circulating DCs, and lower expression levels of HLA-DR, CD123 or CD11c on DCs is associated with fatal COVID-19.
© 2022 Hasan et al.

Podocalyxin (PODXL), a cell surface sialomucin expressed in diverse types of normal and malignant cells, mediates cellular adhesion to extracellular matrix and cell-to-cell interaction. A previous study reported the expression of PODXL protein on monocytes undergoing macrophage differentiation, yet the expression of this molecule in other antigen presenting cells (APCs) and its function in the immune system still remain undetermined. In this study, we report that PODXL is expressed in human monocyte-derived immature dendritic cells at both the mRNA and protein levels. Following dendritric cells maturation using pro-inflammatory stimuli, PODXL expression level decreased substantially. Furthermore, we found that PODXL expression is positively regulated by IL-4 through MEK/ERK and JAK3/STAT6 signaling pathways. Our results revealed a polarized distribution of PODXL during the interaction of APCs with CD4+ T cells, partially colocalizing with F-actin. Notably, PODXL overexpression in APCs promoted their interaction with CD4+ T cells and CD8+ T cells and decreased the expression of MHC-I, MHC-II, and the costimulatory molecule CD86. In addition, PODXL reduced the translocation of CD4+ T-cell centrosome toward the APC-contact site. These findings suggest a regulatory role for PODXL expressed by APCs in immune responses, thus representing a potential target for therapeutic blockade in infection and cancer.
Copyright © 2022 Amo, Díez-García, Tamayo-Orbegozo, Maruri and Larrucea.

The difficulty to unambiguously identify the various subsets of mononuclear phagocytes (MNPs) of the intestinal lamina propria has hindered our understanding of the initial events occurring after mucosal exposure to HIV-1. Here, we compared the composition and function of MNP subsets at steady-state and following ex vivo and in vivo viral exposure in human and macaque colorectal tissues. Combined evaluation of CD11c, CD64, CD103, and CX3CR1 expression allowed to differentiate lamina propria MNPs subsets common to both species. Among them, CD11c+ CX3CR1+ cells expressing CCR5 migrated inside the epithelium following ex vivo and in vivo exposure of colonic tissue to HIV-1 or SIV. In addition, the predominant population of CX3CR1high macrophages present at steady-state partially shifted to CX3CR1low macrophages as early as three days following in vivo SIV rectal challenge of macaques. Our analysis identifies CX3CR1+ MNPs as novel players in the early events of HIV-1 and SIV colorectal transmission.
© 2022 The Author(s).