Sunlnlal'yBipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR)ix[3 and TCR~/8 cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the y and [3 chains of the IL-2 receptor (IL-2Ry and IL-2R[B) and of a specific ix chain (IL-15Rix). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R[B-expressing cells were present in the fetal thymus with a CD25-CD44+FcyR+HSA-/l~ -phenotype, which is characteristic of progenitor ceils. These cells also expressed IL-15Rix messenger RNA. Sorted IL-2R[B+TCR -cells differentiated into TCRixlB and TCR',/8 cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R[~+TCR -cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR',/8 compartment. In contrast, low concentrations oflL-2 did not result in a higher total cell number and did not induce outgrowth of TCRy8 cells. High concentrations of IL-15 blocked TCRix[~ development and shifted differentiation towards NK cells. Differentiation towards TCR2r cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2Rix in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2Rix negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFNγ and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFNγ levels correlate with granzyme B production in CD16+ NK cells and TIM3 expression on CD38+/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCRβ repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3+/CD38+/HLA-DR+ T cells. Using NicheNet, we confirm IFNγ as a central cytokine in the communication between TIM3+/CD38+/HLA-DR+ T cells, CD16+ NK cells, and patrolling monocytes. Normalization of IFNγ, loss of TIM3, quiescence of CD16+ NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis.

In this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine Vγ3 T cell development and activity is assessed. Compared with wild-type (WT) mice, reduced numbers of mature Vγ3 cells were found in the fetal thymus of IL-15−/− mice, while IRF-1−/− mice displayed normal frequencies. Vγ3+ dendritic epidermal T cells (DETCs) were absent in IL-15−/− mice but present in IRF-1−/− mice. DETCs from IRF-1−/− mice displayed morphologically a less mature phenotype and showed different emergence kinetics during ontogeny. This corresponded with lower IL-15 mRNA levels in the skin epidermis. Comparable levels of IL-7 were found in the skin of WT and IL-15−/− mice. Adoptive transfer experiments of WT fetal thymocytes into IL-15−/− mice did not result in the development of Vγ3+ DETCs, confirming the nonredundant role of IL-15 in the skin during DETC development. In vitro, cytolytic activity of IL-15−/− Vγ3 cells was normal after stimulation with IL-15 and was further enhanced by addition of IL-12. In contrast, cytolytic activity of IRF-1−/− Vγ3 cells remained defective after stimulation with IL-15 in combination with IL-12. These data suggest that IL-15 is redundant for the development and/or survival of mature Vγ3 cells in the fetal thymus, whereas it is essential for the localization of Vγ3 cells in the skin. Furthermore, a possible role for IRF-1 in inducing morphological maturation of DETCs and cytolytic capacity of Vγ3 cells is suggested.

Murine NK cells express inhibitory receptors belonging to the Ly49 and CD94/NKG2 family. Ly49E and CD94 are the only NK cell receptor transcripts detectable in fetal NK cells. Still unproved is the surface expression of Ly49E on NK cells. Here we generated two novel mAbs, a mAb recognizing Ly49E with cross-reactivity to Ly49C, and a mAb against NKG2A/C/E. Ly49E was immunoprecipitated as a disulfide-linked homodimer with 46-kDa subunits. Removal of N-linked carbohydrates revealed a 31-kDa protein backbone. NKG2A was immunoprecipitated as a 38-kDa protein. Although the frequency of fetal NK cells expressing Ly49E was higher than 25%, it decreased drastically from 2 wk after birth. Phenotypic analysis showed that ∼90% of fetal NK cells and ∼50% of adult NK cells express high levels of CD94/NKG2. The remaining 50% of adult NK cells expressed low surface levels of CD94/NKG2. Expression of Ly49E and CD94/NKG2 was not restricted to NK cells, but was also observed on NK T and memory T cells. Functional analysis showed that sorted Ly49E+ and CD94/NKG2+ fetal NK cells could discriminate between MHC class I-positive and MHC class I-negative tumor cells. We also demonstrated that Ly49E becomes phosphorylated following pervanadate stimulation of fetal NK cells. The expression levels of Ly49E and CD94/NKG2 were similar in wild-type compared with β2-microglobulin−/− mice. In conclusion, generation of mAbs against Ly49E and NKG2 extended the phenotypic and functional characterization of NK cells.

Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR αβ lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vγ3+ cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rβ (CD122), and absence of IL-2Rα (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located Vγ3 T cells, the progeny of fetal thymic Vγ3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E+ or CD94/NKG2+ Vγ3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vγ3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1b-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vγ3 thymocytes were prevented from killing FcγR+ P815 target cells. These effects were most pronounced in the CD94/NKG2high subpopulation as compared with the CD94/NKG2low subpopulation of Vγ3 cells. Our data demonstrate that Vγ3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.

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Using a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by an intrinsic different NK differentiation potential of fetal versus adult lymphoid progenitors, by immaturity of fetal NK cells or by instability of Ly49E expression. Here, we show that adult progenitor cells were still capable of differentiating into Ly49E-expressing NK cells but at a much lower frequency. Surprisingly, Ly49E expression in vitro did not require stromal cells. Kinetic analysis in vivo showed that Ly49E was expressed early, together with CD94/NKG2 and Ly49G2, followed by Ly49C, and finally Ly49D. Transfer of sorted Ly49E-positive fetal NK cells showed stable Ly49E expression, and later, part of these cells up-regulated other Ly49 members. These data indicate that although there are intrinsic differences, there is no strict fetal and adult wave of NK cell differentiation.

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