Age-Dependent Signature of Serum Inflammatory Cytokines in Healthy Children and Young Adults.

The study of sensitive and specific biomarkers, such as blood inflammatory cytokines, could provide an answer to the challenges faced in the differential diagnosis of patients with systemic inflammation. Limited data exist on the impact of age on serum levels of inflammatory cytokines. We collected serum samples of 42 healthy children and young adults (1 month to 21 years). Serum levels of interleukin 1 receptor antagonist (IL-1Ra), IL-1ß, IL-6, IL-18, tumor necrosis factor-alpha (TNF-α), CXCL9, and CXCL10 were measured. Data were analyzed for three different age groups (<6, 6-17, and 18-21 years). IL-18, TNF-α, and CXCL9 values varied significantly according to age group. Median values of IL-18 and TNF-α decline with age, whereas CXCL9 and CXCL10 are lowest at 6-17 years. IL-1Ra is stable among age groups. In the majority of cases, IL-1ß and IL-6 are not measurable above the lower limit of quantification. A scoping literature review revealed highly variable data on IL-1Ra, IL-18, TNF-α, and CXCL10. For CXCL9, pediatric reference data are scarce. In conclusion, we report an age-dependent signature of multiple inflammatory cytokines measured in the serum of healthy children and young adults, suggesting the need to use age-specific reference values in future pediatric studies.

GTF3A mutations predispose to herpes simplex encephalitis by disrupting biogenesis of the host-derived RIG-I ligand RNA5SP141.

Herpes simplex virus 1 (HSV-1) infects several billion people worldwide and can cause life-threatening herpes simplex encephalitis (HSE) in some patients. Monogenic defects in components of the type I interferon system have been identified in patients with HSE, emphasizing the role of inborn errors of immunity underlying HSE pathogenesis. Here, we identify compound heterozygous loss-of-function mutations in the gene GTF3A encoding for transcription factor IIIA (TFIIIA), a component of the RNA polymerase III complex, in a patient with common variable immunodeficiency and HSE. Patient fibroblasts and GTF3A gene-edited cells displayed impaired HSV-1-induced innate immune responses and enhanced HSV-1 replication. Chromatin immunoprecipitation sequencing analysis identified the 5S ribosomal RNA pseudogene 141 (RNA5SP141), an endogenous ligand of the RNA sensor RIG-I, as a transcriptional target of TFIIIA. GTF3A mutant cells exhibited diminished RNA5SP141 expression and abrogated RIG-I activation upon HSV-1 infection. Our work unveils a crucial role for TFIIIA in transcriptional regulation of a cellular RIG-I agonist and shows that GTF3A genetic defects lead to impaired cell-intrinsic anti-HSV-1 responses and can predispose to HSE.

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C.

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 TCRß 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.

The immunophenotypic fingerprint of patients with primary antibody deficiencies is partially present in their asymptomatic first-degree relatives.

The etiology of primary antibody deficiencies is largely unknown. Beside rare monogenic forms, the majority of cases seem to have a more complex genetic basis. Whereas common variable immunodeficiency has been investigated in depth, there are only a few reports on milder primary antibody deficiencies such as idiopathic primary hypogammaglobulinemia and IgG subclass deficiency. We performed flow cytometric immunophenotyping in 33 patients with common variable immunodeficiency, 23 with idiopathic primary hypogammaglobulinemia and 21 with IgG subclass deficiency, as well as in 47 asymptomatic first-degree family members of patients and 101 unrelated healthy controls. All three groups of patients showed decreased memory B- and naïve T-cell subsets and decreased B-cell activating factor receptor expression. In contrast, circulating follicular helper T-cell frequency and expression of inducible T-cell co-stimulator and chemokine receptors were only significantly altered in patients with common variable immunodeficiency. Asymptomatic first-degree family members of patients demonstrated similar, albeit intermediate, alterations in naïve and memory B- and T-cell subsets. About 13% of asymptomatic relatives had an abnormal peripheral B-cell composition. Furthermore, asymptomatic relatives showed decreased levels of CD4+ recent thymic emigrants and increased central memory T cells. Serum IgG and IgM levels were also significantly lower in asymptomatic relatives than in healthy controls. We conclude that, in our cohort, the immunophenotypic landscape of primary antibody deficiencies comprises a spectrum, in which some alterations are shared between all primary antibody deficiencies whereas others are only associated with common variable immunodeficiency. Importantly, asymptomatic first-degree family members of patients were found to have an intermediate phenotype for peripheral B- and T-cell subsets.

Ly49E-dependent inhibition of natural killer cells by urokinase plasminogen activator.

The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.

Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin-beta receptor expression.

A crucial step in murine natural killer (NK) cell development, mediated by bone marrow stromal cells, is the induction of Ly49 and CD94/NKG2 receptor expression. The signals that regulate Ly49 receptor expression are still largely undetermined. It has been shown that interaction between lymphotoxin alpha1beta2 (LTalpha1beta2) and LTbeta receptor (LTbetaR), expressed on lymphoid progenitor cells and nonlymphoid bone marrow stromal cells, respectively, is important for both quantitative and functional NK cell development. Therefore, we have investigated the role of LT-LTbetaR-mediated signaling in Ly49 and CD94/NKG2 receptor acquisition. We show that the NK receptor repertoire of LTbetaR-/- mice can only be partially analyzed because of the residual 129/Ola mouse genetic background, due to a physical linkage of the LTbetaR locus and the loci encoding the Ly49 and CD94/NKG2 receptors. Therefore, we transferred wild-type B6 lymphoid-committed progenitor cells into LTbetaR-/- mice, which differentiated into NK cells with a normal NK cell receptor repertoire. Also, administration of LTbetaR-immunoglobulin (Ig), which acts as a soluble receptor for LTalpha1beta2, resulted in reduced NK cell percentages but did not influence the Ly49 and CD94/NKG2 receptor acquisition on remaining NK cells. These results indicate that LTbetaR-mediated signals are not required for Ly49 and CD94/NKG2 receptor acquisition.

Role of natural killer cells in the rejection process of corneal allografts in rats.

BACKGROUND: The exact mechanism of human corneal allograft rejection, which is the major cause of corneal transplant failure, remains unclear. We investigated the role of natural killer (NK) cells in rat corneal allograft rejection by examining the aqueous humor (AH) cell infiltrate on different postoperative days. METHODS: Flow cytometric analysis was performed on the AH and submandibular draining lymph node (DLN) cells before transplantation and at different time points thereafter. In addition, we performed functional cytotoxicity assays with cells present in the AH during corneal rejection. RESULTS: We demonstrated a gradual increase in the absolute cell number of different hematopoietic subpopulations in the AH after allogeneic cornea transplantation. CD3CD4 cells, mainly monocytes and macrophages, were the predominant subpopulation 2 days after transplantation, followed by a successive relative increase of CD4 T cells, CD8 T cells, CD161 T cells, and NK cells. NK and CD161 T cells were present at a 10- to 15-fold higher percentage than in the DLN, suggestive of local expansion of these cells. A higher percentage of NK cells were CD8-negative compared with DLN NK cells. AH cells specifically lysed allogeneic cells, and this cytotoxicity was mainly attributable to NK cells but not to CD4 or CD8 T lymphocytes. CONCLUSION: These results confirm the crucial role of CD4 cells in the allogeneic corneal graft rejection process and implicate NK cells as possible mediators of the rejection.

Ly49E expression points toward overlapping, but distinct, natural killer (NK) cell differentiation kinetics and potential of fetal versus adult lymphoid progenitors.

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.

Developmental and functional defects of thymic and epidermal V gamma 3 cells in IL-15-deficient and IFN regulatory factor-1-deficient mice.

In this study, the role of IL-15 and its regulation by the transcription factor IFN regulatory factor-1 (IRF-1) in murine V gamma 3 T cell development and activity is assessed. Compared with wild-type (WT) mice, reduced numbers of mature V gamma 3 cells were found in the fetal thymus of IL-15(-/-) mice, while IRF-1(-/-) mice displayed normal frequencies. V gamma 3(+) 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 V gamma 3(+) DETCs, confirming the nonredundant role of IL-15 in the skin during DETC development. In vitro, cytolytic activity of IL-15(-/-) V gamma 3 cells was normal after stimulation with IL-15 and was further enhanced by addition of IL-12. In contrast, cytolytic activity of IRF-1(-/-) V gamma 3 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 V gamma 3 cells in the fetal thymus, whereas it is essential for the localization of V gamma 3 cells in the skin. Furthermore, a possible role for IRF-1 in inducing morphological maturation of DETCs and cytolytic capacity of V gamma 3 cells is suggested.

Expression of inhibitory receptors Ly49E and CD94/NKG2 on fetal thymic and adult epidermal TCR V gamma 3 lymphocytes.

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 alphabeta lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR Vgamma3(+) cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2Rbeta (CD122), and absence of IL-2Ralpha (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 Vgamma3 T cells, the progeny of fetal thymic Vgamma3 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(+) Vgamma3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR Vgamma3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1(b)-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR Vgamma3 thymocytes were prevented from killing FcgammaR(+) P815 target cells. These effects were most pronounced in the CD94/NKG2(high) subpopulation as compared with the CD94/NKG2(low) subpopulation of Vgamma3 cells. Our data demonstrate that Vgamma3 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.