Characterization of cord blood CD3+ TCRVα7.2+ CD161high T and innate lymphoid cells in the pregnancies with gestational diabetes, morbidly adherent placenta, and pregnancy hypertension diseases.

PROBLEM: Although pregnant women with gestational diabetes (GD), morbidly adherent placenta (MAP), and pregnancy hypertension (pHT) diseases lead to intrauterine growth restriction (IUGR), little is known about their effect on mucosal-associated invariant T (MAIT) and innate lymphoid cells (ILC) in the umbilical cord. This study aimed to quantify and characterize MAIT cells and ILCs in the cord blood of pregnant women with GD, MAP, and pHT diseases. METHOD OF STUDY: Cord blood mononuclear cells (CBMCs) were isolated by Ficoll-Paque gradient. CD3+ TCRVα7.2+ CD161high cells and ILC subsets were quantified by flow cytometry. CBMCs were stimulated with PMA/Ionomycin and Golgi Plug for 4 h and stained for IFN-γ, TNF-α, and granzyme B. The stained cells were analyzed on FACS ARIA III. RESULTS: Compared with healthy pregnancies, in the cord blood of the pHT group, elevated number of lymphocytes was observed. Moreover, the absolute number of IFN-γ producing CD4+ or CD4- subsets of CD3+ TCRVα7.2+ CD161high cells as well as those producing granzyme B were significantly elevated in the pHT group compared to healthy controls suggesting increased MAIT cell activity in the pHT cord blood. Similarly, in the MAP group, the absolute number of total CD3+ TCRVα7.2+ CD161high cells, but not individual CD4+ or negative subsets, were significantly increased compared with healthy controls' cord blood. Absolute numbers of total CD3+ TCRVα7.2+ CD161high cells and their subsets were comparable in the cord blood of the GD group compared with healthy controls. Finally, the absolute number of total ILCs and ILC3 subset were significantly elevated in only pHT cord blood compared with healthy controls. Our data also reveal that IFN-γ+ or granzyme B+ cell numbers negatively correlated with fetal birth weight. CONCLUSIONS: CD3+ TCRVα7.2+ CD161high cells and ILCs show unique expansion and activity in the cord blood of pregnant women with distinct diseases causing IUGR and may play roles in fetal growth restriction.

ILC3 deficiency and generalized ILC abnormalities in DOCK8-deficient patients.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is the main cause of the autosomal recessive hyper-IgE syndrome (HIES). We previously reported the selective loss of group 3 innate lymphoid cell (ILC) number and function in a Dock8-deficient mouse model. In this study, we sought to test whether DOCK8 is required for the function and maintenance of ILC subsets in humans. METHODS: Peripheral blood ILC1-3 subsets of 16 DOCK8-deficient patients recruited at the pretransplant stage, and seven patients with autosomal dominant (AD) HIES due to STAT3 mutations, were compared with those of healthy controls or post-transplant DOCK8-deficient patients (n = 12) by flow cytometry and real-time qPCR. Sorted total ILCs from DOCK8- or STAT3-mutant patients and healthy controls were assayed for survival, apoptosis, proliferation, and activation by IL-7, IL-23, and IL-12 by cell culture, flow cytometry, and phospho-flow assays. RESULTS: DOCK8-deficient but not STAT3-mutant patients exhibited a profound depletion of ILC3s, and to a lesser extent ILC2s, in their peripheral blood. DOCK8-deficient ILC1-3 subsets had defective proliferation, expressed lower levels of IL-7R, responded less to IL-7, IL-12, or IL-23 cytokines, and were more prone to apoptosis compared with those of healthy controls. CONCLUSION: DOCK8 regulates human ILC3 expansion and survival, and more globally ILC cytokine signaling and proliferation. DOCK8 deficiency leads to loss of ILC3 from peripheral blood. ILC3 deficiency may contribute to the susceptibility of DOCK8-deficient patients to infections.

Genetic Deficiency and Biochemical Inhibition of ITK Affect Human Th17, Treg, and Innate Lymphoid Cells.

PURPOSE: Interleukin-2-inducible T cell kinase (ITK) is an important mediator of T cell receptor signaling. Loss of function mutations in ITK results in hypogammaglobulinemia and CD4+ T cell loss in humans, and the patients often present with EBV-associated B cell lymphoproliferative syndrome. Itk-deficient mice show loss of T cell naivety, impaired cytolytic activity of CD8+ T cells, and defects in CD4+ T cell lineage choice decisions. In mice, Itk mutations were shown to affect Th17-Treg lineage choice in favor of the latter. In this study, we explored whether human ITK reciprocally regulates Th17-Treg balance as its murine ortholog. METHODS: Whole Exome Sequencing was used to identify the mutation. ITK-deficient peripheral blood lymphocytes were characterized by FACSAria III-based flow cytometric assays with respect to proliferation, apoptosis, cytokine production, and innate lymphoid cell (ILC) frequency. Sorted T cells from healthy donors were exposed to ibrutinib, an irreversible ITK inhibitor, to assess ITK's contribution to Th17 and Treg cell generation and functions. RESULTS: In this study, we report a child with a novel ITK mutation who showed impaired CD3/CD28 induced proliferation in T cells. ITK-mutant cells were more apoptotic irrespective of TCR activation. More importantly, T cells produced less Th17-associated cytokines IL-17A, IL-22, and GM-CSF. Conversely, Th1-associated IFN-γ production was increased. An irreversible inhibitor of ITK, ibrutinib, blocked ex vivo Th17 generation and IL-17A production, conversely augmented FOXP3 expression only at low doses in Treg cultures. Finally, we analyzed peripheral ILC populations and observed a relative decrease in ILC2 and ILC3 frequency in our ITK-deficient patient. CONCLUSIONS: To our knowledge, this is the first report showing that both genetic and chemical inhibition of ITK result in reduced Th17 generation and function in humans. We also report, for the first time, a reduction in ILC2 and ILC3 populations in an ITK-deficient human patient.

Fingolimod Alters Tissue Distribution and Cytokine Production of Human and Murine Innate Lymphoid Cells.

Sphingosine-1 phosphate receptor 1 (S1PR1) is expressed by lymphocytes and regulates their egress from secondary lymphoid organs. Innate lymphoid cell (ILC) family has been expanded with the discovery of group 1, 2 and 3 ILCs, namely ILC1, ILC2 and ILC3. ILC3 and ILC1 have remarkable similarity to CD4+ helper T cell lineage members Th17 and Th1, respectively, which are important in the pathology of multiple sclerosis (MS). Whether human ILC subsets express S1PR1 or respond to its ligands have not been studied. In this study, we used peripheral blood/cord blood and tonsil lymphocytes as a source of human ILCs. We show that human ILCs express S1PR1 mRNA and protein and migrate toward S1P receptor ligands. Comparison of peripheral blood ILC numbers between fingolimod-receiving and treatment-free MS patients revealed that, in vivo, ILCs respond to fingolimod, an S1PR1 agonist, resulting in ILC-penia in circulation. Similarly, murine ILCs responded to fingolimod by exiting blood and accumulating in the secondary lymph nodes. Importantly, ex vivo exposure of ILC3 and ILC1 to fingolimod or SEW2871, another S1PR1 antagonist, reduced production of ILC3- and ILC1- associated cytokines GM-CSF, IL-22, IL-17, and IFN-γ, respectively. Surprisingly, despite reduced number of lamina propria-resident ILC3s in the long-term fingolimod-treated mice, ILC3-associated IL-22, IL-17A, GM-CSF and antimicrobial peptides were high in the gut compared to controls, suggesting that its long term use may not compromise mucosal barrier function. To our knowledge, this is the first study to investigate the impact of fingolimod on human ILC subsets in vivo and ex vivo, and provides insight into the impact of long term fingolimod use on ILC populations.