Proteogenomic and V(D)J Analysis of Human Decidual T Cells Highlights Unique Transcriptional Programming and Clonal Distribution.

Immunological tolerance toward the semiallogeneic fetus is one of many maternal adaptations required for a successful pregnancy. T cells are major players of the adaptive immune system and balance tolerance and protection at the maternal-fetal interface; however, their repertoire and subset programming are still poorly understood. Using emerging single-cell RNA sequencing technologies, we simultaneously obtained transcript, limited protein, and receptor repertoire at the single-cell level, from decidual and matched maternal peripheral human T cells. The decidua maintains a tissue-specific distribution of T cell subsets compared with the periphery. We find that decidual T cells maintain a unique transcriptome programming, characterized by restraint of inflammatory pathways by overexpression of negative regulators (DUSP, TNFAIP3, ZFP36) and expression of PD-1, CTLA-4, TIGIT, and LAG3 in some CD8 clusters. Finally, analyzing TCR clonotypes demonstrated decreased diversity in specific decidual T cell populations. Overall, our data demonstrate the power of multiomics analysis in revealing regulation of fetal-maternal immune coexistence.

Cotyledon-Specific Flow Evaluation of Rhesus Macaque Placental Injury Using Ferumoxytol Dynamic Contrast-Enhanced MRI.

BACKGROUND: Recently, dynamic contrast-enhanced (DCE) MRI with ferumoxytol as contrast agent has recently been introduced for the noninvasive assessment of placental structure and function throughout. However, it has not been demonstrated under pathological conditions. PURPOSE: To measure cotyledon-specific rhesus macaque maternal placental blood flow using ferumoxytol DCE MRI in a novel animal model for local placental injury. STUDY TYPE: Prospective animal model. SUBJECTS: Placental injections of Tisseel (three with 0.5 mL and two with 1.5 mL), monocyte chemoattractant protein 1 (three with 100 µg), and three with saline as controls were performed in a total of 11 rhesus macaque pregnancies at approximate gestational day (GD 101). DCE MRI scans were performed prior (GD 100) and after (GD 115 and GD 145) the injection (term = GD 165). FIELD STRENGTH/SEQUENCE: 3 T, T1-weighted spoiled gradient echo sequence (product sequence, DISCO). ASSESSMENT: Source images were inspected for motion artefacts from the mother or fetus. Placenta segmentation and DCE processing were performed for the dynamic image series to measure cotyledon specific volume, flow, and normalized flow. Overall placental histopathology was conducted for controls, Tisseel, and MCP-1 animals and regions of tissue infarctions and necrosis were documented. Visual inspections for potential necrotic tissue were conducted for the two Tisseelx3 animals. STATISTICAL TESTS: Wilcoxon rank sum test, significance level P < 0.05. RESULTS: No motion artefacts were observed. For the group treated with 1.5 mL of Tisseel, significantly lower cotyledon volume, flow, and normalized flow per cotyledon were observed for the third gestational time point of imaging (day ~145), with mean normalized flow of 0.53 minute-1. Preliminary histopathological analysis shows areas of tissue necrosis from a selected cotyledon in one Tisseel-treated (single dose) animal and both Tisseelx3 (triple dose) animals. DATA CONCLUSION: This study demonstrates the feasibility of cotyledon-specific functional analysis at multiple gestational time points and injury detection in a placental rhesus macaque model through ferumoxytol-enhanced DCE MRI. LEVEL OF EVIDENCE: NA TECHNICAL EFFICACY: Stage 2.

Continuous positive airway pressure treatment of obstructive sleep apnea and hypertensive complications in high-risk pregnancy.

PURPOSE: To evaluate whether or not continuous positive airway pressure (CPAP) treatment in pregnancies complicated by obstructive sleep apnea (OSA) is associated with a decrease in hypertensive disorders of pregnancy. METHODS: This was a retrospective cohort study of perinatal outcomes in women who underwent objective OSA testing and treatment as part of routine clinical care during pregnancy. Where diagnostic criteria for OSA were reached (respiratory event index (REI) ≥ 5 events per hour), patients were offered CPAP therapy. Obstetrical outcomes were compared between the control group (no OSA), the group with untreated OSA (OSA diagnosed, not CPAP compliant), and the group with treated OSA (OSA diagnosed and CPAP compliant), with CPAP compliance defined as CPAP use ≥ 4 h, 70% of the time or greater. A composite hypertension outcome combined diagnoses of gestational hypertension (gHTN) and preeclampsia (PreE) of any severity. RESULTS: The study comprised outcomes from 177 completed pregnancies. Our cohort was characterized by obesity, with average body mass indices > 35 kg/m2, and average maternal age > 30 years old. CPAP was initiated at an average gestational age of 23 weeks (12.1-35.3 weeks), and average CPAP use was 5.9 h (4-8.5 h). The composite hypertension outcome occurred in 43% of those without OSA (N = 77), 64% of those with untreated OSA (N = 77), and 57% of those with treated OSA, compliant with CPAP (N = 23) (p = 0.034). CONCLUSION: Real-world data in this small study suggest that CPAP therapy may modulate the increased risk of hypertensive complications in pregnancies complicated by OSA.

The promise of placental extracellular vesicles: models and challenges for diagnosing placental dysfunction in utero†.

Monitoring the health of a pregnancy is of utmost importance to both the fetus and the mother. The diagnosis of pregnancy complications typically occurs after the manifestation of symptoms, and limited preventative measures or effective treatments are available. Traditionally, pregnancy health is evaluated by analyzing maternal serum hormone levels, genetic testing, ultrasonographic imaging, and monitoring maternal symptoms. However, researchers have reported a difference in extracellular vesicle (EV) quantity and cargo between healthy and at-risk pregnancies. Thus, placental EVs (PEVs) may help to understand normal and aberrant placental development, monitor pregnancy health in terms of developing placental pathologies, and assess the impact of environmental influences, such as infection, on pregnancy. The diagnostic potential of PEVs could allow for earlier detection of pregnancy complications via noninvasive sampling and frequent monitoring. Understanding how PEVs serve as a means of communication with maternal cells and recognizing their potential utility as a readout of placental health have sparked a growing interest in basic and translational research. However, to date, PEV research with animal models lags behind human studies. The strength of animal pregnancy models is that they can be used to assess placental pathologies in conjunction with isolation of PEVs from fluid samples at different time points throughout gestation. Assessing PEV cargo in animals within normal and complicated pregnancies will accelerate the translation of PEV analysis into the clinic for potential use in prognostics. We propose that appropriate animal models of human pregnancy complications must be established in the PEV field.

Comparison of γ-Aminobutyric Acid, Type A (GABAA), Receptor αßγ and αßδ Expression Using Flow Cytometry and Electrophysiology: EVIDENCE FOR ALTERNATIVE SUBUNIT STOICHIOMETRIES AND ARRANGEMENTS.

The subunit stoichiometry and arrangement of synaptic αßγ GABAA receptors are generally accepted as 2α:2ß:1γ with a ß-α-γ-ß-α counterclockwise configuration, respectively. Whether extrasynaptic αßδ receptors adopt the analogous ß-α-δ-ß-α subunit configuration remains controversial. Using flow cytometry, we evaluated expression levels of human recombinant γ2 and δ subunits when co-transfected with α1 and/or ß2 subunits in HEK293T cells. Nearly identical patterns of γ2 and δ subunit expression were observed as follows: both required co-transfection with α1 and ß2 subunits for maximal expression; both were incorporated into receptors primarily at the expense of ß2 subunits; and both yielded similar FRET profiles when probed for subunit adjacency, suggesting similar underlying subunit arrangements. However, because of a slower rate of δ subunit degradation, 10-fold less δ subunit cDNA was required to recapitulate γ2 subunit expression patterns and to eliminate the functional signature of α1ß2 receptors. Interestingly, titrating γ2 or δ subunit cDNA levels progressively altered GABA-evoked currents, revealing more than one kinetic profile for both αßγ and αßδ receptors. This raised the possibility of alternative receptor isoforms, a hypothesis confirmed using concatameric constructs for αßγ receptors. Taken together, our results suggest a limited cohort of alternative subunit arrangements in addition to canonical ß-α-γ/δ-ß-α receptors, including ß-α-γ/δ-α-α receptors at lower levels of γ2/δ expression and ß-α-γ/δ-α-γ/δ receptors at higher levels of expression. These findings provide important insight into the role of GABAA receptor subunit under- or overexpression in disease states such as genetic epilepsies.

Association between peak estradiol levels and ovarian torsion among symptomatic patients receiving gonadotropin treatment.

PURPOSE: Ovarian torsion is a surgical emergency that can be clinically challenging to diagnose. Patients who have received assisted reproductive technologies (ART) are a subset of women with an increased risk for torsion. As the ART population continues to increase, there is a need to delineate risk factors for the development of ovarian torsion in this unique population. A pilot study was performed to determine the proportion of patients with suspected ovarian torsion who have received ART and to identify possible diagnostic biomarkers for ovarian torsion among these patients. METHODS: A single institution retrospective cohort study of patients taken to surgery for suspected ovarian torsion over a 5-year period. RESULTS: During the study period, 171 patients were taken to surgery for suspected ovarian torsion. Patients receiving ART constituted 19 (11%) of these patients. Among the 19 fertility treatment patients, 16 had received treatment with gonadotropins, 10 of which had surgically confirmed ovarian torsion. These ten patients had higher preoperative peak estradiol levels (3122 versus 1875 pg/mL, p = 0.05) and a larger ovarian diameter (9.7 versus 7.6 cm, p = 0.05) than the six patients receiving gonadotropins found to not have ovarian torsion. CONCLUSIONS: These results suggest infertility treatment using gonadotropins for ovarian hyperstimulation may be an independent risk factor for ovarian torsion as suggested by the disproportionate number of such individuals represented in the study population (9% of all patients, 84% of fertility patients). Additionally, among women taking gonadotropins, an association exists between peak estradiol levels, ovarian diameter, and risk for ovarian torsion.

GABAA receptor biogenesis is impaired by the γ2 subunit febrile seizure-associated mutation, GABRG2(R177G).

A missense mutation in the GABAA receptor γ2L subunit, R177G, was reported in a family with complex febrile seizures (FS). To gain insight into the mechanistic basis for these genetic seizures, we explored how the R177G mutation altered the properties of recombinant α1ß2γ2L GABAA receptors expressed in HEK293T cells. Using a combination of electrophysiology, flow cytometry, and immunoblotting, we found that the R177G mutation decreased GABA-evoked whole-cell current amplitudes by decreasing cell surface expression of α1ß2γ2L receptors. This loss of receptor surface expression resulted from endoplasmic reticulum (ER) retention of mutant γ2L(R177G) subunits, which unlike wild-type γ2L subunits, were degraded by ER-associated degradation (ERAD). Interestingly, when compared to the condition of homozygous γ2L(R177G) subunit expression, disproportionately low levels of γ2L(R177G) subunits reached the cell surface with heterozygous expression, indicating that wild-type γ2L subunits possessed a competitive advantage over mutant γ2L(R177G) subunits for receptor assembly and/or forward trafficking. Inhibiting protein synthesis with cycloheximide demonstrated that the R177G mutation primarily decreased the stability of an intracellular pool of unassembled γ2L subunits, suggesting that the mutant γ2L(R177G) subunits competed poorly with wild-type γ2L subunits due to impaired subunit folding and/or oligomerization. Molecular modeling confirmed that the R177G mutation could disrupt intrasubunit salt bridges, thereby destabilizing secondary and tertiary structure of γ2L(R177G) subunits. These findings support an emerging body of literature implicating defects in GABAA receptor biogenesis in the pathogenesis of genetic epilepsies (GEs) and FS.

Longitudinal expression of Toll-like receptors on dendritic cells in uncomplicated pregnancy and postpartum.

OBJECTIVE: Toll-like receptors (TLRs) are integral parts of the innate immune system and have been implicated in complications of pregnancy. The longitudinal expression of TLRs on dendritic cells in the maternal circulation during uncomplicated pregnancies is unknown. The objective of this study was to prospectively evaluate TLRs 1-9 as expressed on dendritic cells in the maternal circulation at defined intervals throughout pregnancy and postpartum. STUDY DESIGN: This was a prospective cohort of 30 pregnant women with uncomplicated pregnancies and 30 nonpregnant controls. TLRs and cytokine expression was measured in unstimulated dendritic cells at 4 defined intervals during pregnancy and postpartum. Basal expression of TLRs and cytokines was measured by multicolor flow cytometry. The percent-positive dendritic cells for each TLRs were compared with both nonpregnant and postpartum levels with multivariate linear regression. RESULTS: TLRs 1, 7, and 9 were elevated compared with nonpregnant controls with persistent elevation of TLR 1 and interleukin-12 (IL-12) into the postpartum period. Concordantly, levels of IL-6, IL-12, interferon alpha, and tumor necrosis factor alpha increased during pregnancy and returned to levels similar to nonpregnant controls during the postpartum period. The elevated levels of TLR 1 and IL-12 were persistent postpartum, challenging notions that immunologic changes during pregnancy resolve after the prototypical postpartum period. CONCLUSION: Normal pregnancy is associated with time-dependent changes in TLR expression compared with nonpregnant controls; these findings may help elucidate immunologic dysfunction in complicated pregnancies.

Decidual leukocytes respond to African lineage Zika virus infection with mild anti-inflammatory changes during acute infection in rhesus macaques.

Zika virus (ZIKV) can be vertically transmitted during pregnancy resulting in a range of adverse pregnancy outcomes. The decidua is commonly found to be infected by ZIKV, yet the acute immune response to infection remains understudied in vivo. We hypothesized that in vivo African-lineage ZIKV infection induces a pro-inflammatory response in the decidua. To test this hypothesis, we evaluated the decidua in pregnant rhesus macaques within the first two weeks following infection with an African-lineage ZIKV and compared our findings to gestationally aged-matched controls. Decidual leukocytes were phenotypically evaluated using spectral flow cytometry, and cytokines and chemokines were measured in tissue homogenates from the decidua, placenta, and fetal membranes. The results of this study did not support our hypothesis. Although ZIKV RNA was detected in the decidual tissue samples from all ZIKV infected dams, phenotypic changes in decidual leukocytes and differences in cytokine profiles suggest that the decidua undergoes mild anti-inflammatory changes in response to that infection. Our findings emphasize the immunological state of the gravid uterus as a relatively immune privileged site that prioritizes tolerance of the fetus over mounting a pro-inflammatory response to clear infection.

Corrigendum: Deciphering decidual leukocyte traffic with serial intravascular staining.

[This corrects the article DOI: 10.3389/fimmu.2023.1332943.].

IL-15 regulates homeostasis and terminal maturation of NKT cells.

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

Uterine fibroids and subfertility: an update on the role of myomectomy.

PURPOSE OF REVIEW: Uterine fibroids, the most common neoplasm of reproductive-aged women, can have a significant impact on quality of life, and may affect fertility and pregnancy outcomes. Although it is generally accepted that submucosal fibroids are of clinical significance, the effect of intramural and subserosal fibroids, and the benefit of surgical removal remains an area of active debate. Because of this controversy, this article will review current evidence for an association of fibroids and subfertility, and assess the impact of surgical management on fertility outcomes. RECENT FINDINGS: Recent analyses of patients with intramural fibroids have reported an increase in pregnancy loss and reduction in pregnancy and live birth rates. However, when analyzing studies with high quality diagnostic methods for assessing the endometrial cavity, no significant impact on reproductive outcomes was observed, and no benefit of myomectomy was consistently demonstrated. Myomectomy for submucosal fibroids greater than 2 cm and for intramural fibroids distorting the endometrial contour likely confers improvement of fertility outcome. SUMMARY: Submucosal fibroid location and distortion of the endometrial cavity (either submucosal or deeply infiltrating intramural fibroids) are most predictive of impaired fertility and probable benefit of surgical removal, and warrant consideration of myomectomy in the subfertile patient.

Immune Dysfunction in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-aged individuals with ovaries. It is associated with anovulation and increased risk to fertility and metabolic, cardiovascular, and psychological health. The pathophysiology of PCOS is still inadequately understood, although there is evidence of persistent low-grade inflammation, which correlates with associated visceral obesity. Elevated proinflammatory cytokine markers and altered immune cells have been reported in PCOS and raise the possibility that immune factors contribute to ovulatory dysfunction. Because normal ovulation is modulated by immune cells and cytokines in the ovarian microenvironment, the endocrine and metabolic abnormalities associated with PCOS orchestrate the accompanying adverse effects on ovulation and implantation. This review evaluates the current literature on the relationship between PCOS and immune abnormalities, with a focus on emerging research in the field.

Mononuclear Cells Negatively Regulate Endothelial Ca2+ Signaling.

Endothelial Ca2+ signaling has important roles to play in maintaining pregnancy associated vasodilation in the utero-placenta. Inflammatory cytokines, often elevated in vascular complications of pregnancy, negatively regulate ATP-stimulated endothelial Ca2+ signaling and associated nitric oxide production. However, the role of direct engagement of immune cells on endothelial Ca2+ signaling and therefore endothelial function is unclear. To model immune-endothelial interactions, herein, we evaluate the effects of peripheral blood mononuclear cells (PBMCs) in short-term interaction with human umbilical vein endothelial cells (HUVECs) on agonist-stimulated Ca2+ signaling in HUVECs. We find that mononuclear cells (10:1 and 25:1 mononuclear: HUVEC) cause decreased ATP-stimulated Ca2+ signaling; worsened by activated mononuclear cells possibly due to increased cytokine secretion. Additionally, monocytes, natural killers, and T-cells cause decrease in ATP-stimulated Ca2+ signaling using THP-1 (monocyte), NKL (natural killer cells), and Jurkat (T-cell) cell lines, respectively. PBMCs with Golgi-restricted protein transport prior to interaction with endothelial cells display rescue in Ca2+ signaling, strongly suggesting that secreted proteins from PBMCs mediate changes in HUVEC Ca2+ signaling. We propose that endothelial cells from normal pregnancy interacting with PBMCs may model preeclamptic endothelial-immune interaction and resultant endothelial dysfunction.

Deciphering decidual leukocyte traffic with serial intravascular staining.

The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, the degree, timing, and subset-specific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques (Macaca fascicularis) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy.

Multiparameter Flow Cytometry for Detailed Characterization of Peritoneal Immune Cells from Patients with Ovarian Cancer.

Multiparameter flow cytometry is a convenient and efficient method for thorough phenotyping of cells, and especially immune cells from various tissues. We have successfully used multiparameter flow cytometry to characterize immune cells from patients with ovarian cancer and leveraged dimensionality reduction and machine learning for optimized visualization and analysis. Herein, we describe our optimized and established protocols for the labeling of cells with fluorophore-conjugated antibody panels, followed by details on data acquisition. Finally, we describe methods for analysis of the flow cytometry data using both FlowJo as well as R package, Cytofkit, for multidimensional data visualization.

Identifying novel ovarian tumor biomarkers through mining of the transcriptome of circulating immune cells: A proof-of-concept study.

OBJECTIVE: Treatment of high-grade serous ovarian cancer (HGSOC) will benefit from early detection of cancer. Here, we provide proof-of-concept data supporting the hypothesis that circulating immune cells, because of their early recognition of tumors and the tumor microenvironment, can be considered for biomarker discovery. METHODS: Longitudinal blood samples from C57BL/6 mice bearing syngeneic ovarian tumors and peripheral blood mononuclear cells (PBMC) from healthy postmenopausal women and newly diagnosed for HGSOC patients were subjected to RNASeq. The results from human immune cells were validated using Affymetrix microarrays. Differentially expressed transcripts in immune cells from tumor-bearing mice and HGSOC patients were compared to matching controls. RESULTS: A total of 1282 transcripts (798 and 484, up- and downregulated, respectively) were differentially expressed in the tumor-bearing mice as compared with controls. Top 100 genes showing longitudinal changes in gene expression 2, 4, 7, and 18 days after tumor implantation were identified. Analysis of the PBMC from healthy post-menopausal women and HGSOC patients identified 4382 differentially expressed genes and 519 of these were validated through Affymetrix microarray analysis. A total of 384 genes, including IL-1R2, CH3L1, Infitm1, FP42, CXC42, Hdc, Spib, and Sema6b, were differentially expressed in the human and mouse datasets. CONCLUSION: The PBMC transcriptome shows longitudinal changes in response to the progressing tumor. Several potential biomarker transcripts were identified in HGSOC patients and mouse models. Monitoring their expression in individual PBMC subsets can serve as additional discriminator for the diagnosis of HGSOC.

Multiomic analysis reveals decidual-specific transcriptional programing of MAIT cells.

PROBLEM: Mucosal-Associated Invariant T (MAIT) cells have been recently identified at the maternal-fetal interface. However, transcriptional programming of decidual MAIT cells in pregnancy remains poorly understood. METHOD OF STUDY: We employed a multiomic approach to address this question. Mononuclear cells from the decidua basalis and parietalis, and control PBMCs, were analyzed via flow cytometry to investigate MAIT cells in the decidua and assess their transcription factor expression. In a separate study, both decidual and matched peripheral MAIT cells were analyzed using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) coupled with gene expression analysis. Lastly, decidual MAIT cells were stimulated with E.coli and expression of MR1 by antigen presenting cells was measured to evaluate decidual MAIT cell function. RESULTS: First, we identified MAIT cells in both the decidua basalis and parietalis. CITE-seq, coupled with scRNA-seq gene expression analysis, highlighted transcriptional programming differences between decidual and matched peripheral MAIT cells at a single cell resolution. Transcription factor expression analysis further highlighted transcriptional differences between decidual MAIT cells and non-matched peripheral MAIT cells. Functionally, MAIT cells are skewed towards IFNγ and TNFα production upon stimulation, with E.coli leading to IFNγ production. Lastly, we demonstrate that MR1, the antigen presenting molecule restricting MAIT cells, is expressed by decidual APCs. CONCLUSION: MAIT cells are present in the decidua basalis and obtain a unique gene expression profile. The presence of MR1 on APCs coupled with in vitro activation by E.coli suggests that MAIT cells might be involved in tissue-repair mechanisms at the maternal-fetal interface.

CD1d-restricted human natural killer T cells are highly susceptible to human immunodeficiency virus 1 infection.

Human natural killer (NK) T cells are unique T lymphocytes that express an invariant T cell receptor (TCR) Valpha24-Vbeta11 and have been implicated to play a role in various diseases. A subset of NKT cells express CD4 and hence are potential targets for human immunodeficiency virus (HIV)-1 infection. We demonstrate that both resting and activated human Valpha24(+) T cells express high levels of the HIV-1 coreceptors CCR5 and Bonzo (CXCR6), but low levels of CCR7, as compared with conventional T cells. Remarkably NKT cells activated with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells were profoundly more susceptible to infection with R5-tropic, but not X4-tropic, strains of HIV-1, compared with conventional CD4(+) T cells. Furthermore, resting CD4(+) NKT cells were also more susceptible to infection. After initial infection, HIV-1 rapidly replicated and depleted the CD4(+) subset of NKT cells. In addition, peripheral blood NKT cells were markedly and selectively depleted in HIV-1 infected individuals. Although the mechanisms of this decline are not clear, low numbers or absence of NKT cells may affect the course of HIV-1 infection. Taken together, our findings indicate that CD4(+) NKT cells are directly targeted by HIV-1 and may have a potential role during viral transmission and spread in vivo.

CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells.

Natural killer T (NKT) cells are a unique immunoregulatory T cell population that is positively selected by CD1d-expressing thymocytes. Previous studies have shown that NKT cells exhibit autoreactivity, which raises the question of whether they are subject to negative selection. Here, we report that the addition of agonist glycolipid alpha-galactosylceramide (alpha-GalCer) to a fetal thymic organ culture (FTOC) induces a dose-dependent disappearance of NKT cells, suggesting that NKT cells are susceptible to negative selection. Overexpression of CD1d in transgenic (Tg) mice results in reduced numbers of NKT cells, and the residual NKT cells in CD1d-Tg mice exhibit both an altered Vbeta usage and a reduced sensitivity to antigen. Furthermore, bone marrow (BM) chimeras between Tg and WT mice reveal that CD1d-expressing BM-derived dendritic cells, but not thymic epithelial cells, mediate the efficient negative selection of NKT cells. Thus, our data suggest that NKT cells developmentally undergo negative selection when engaged by high-avidity antigen or abundant self-antigen.