Frequency Of NKT Cells Research Articles

Mucosal-associated invariant T cell deficiency in systemic lupus erythematosus.

Mucosal-associated invariant T (MAIT) cells contribute to protection against certain microorganism infections and play an important role in mucosal immunity. However, the role of MAIT cells remains enigmatic in autoimmune diseases. In this study, we examined the level and function of MAIT cells in patients with rheumatic diseases. MAIT cell, cytokine, and programmed death-1 (PD-1) levels were measured by flow cytometry. Circulating MAIT cell levels were significantly reduced in systemic lupus erythematosus (SLE) and rheumatoid arthritis patients. In particular, this MAIT cell deficiency was more prominent in CD8(+) and double-negative T cell subsets, and significantly correlated with disease activity, such as SLE disease activity index and 28-joint disease activity score. Interestingly, MAIT cell frequency was significantly correlated with NKT cell frequency in SLE patients. IFN-γ production in MAIT cells was impaired in SLE patients, which was due to an intrinsic defect in the Ca(2+)/calcineurin/NFAT1 signaling pathway. In SLE patients, MAIT cells were poorly activated by α-galactosylceramide-stimulated NKT cells, thereby showing the dysfunction between MAIT cells and NKT cells. Notably, an elevated expression of PD-1 in MAIT cells and NKT cells was associated with SLE. In rheumatoid arthritis patients, MAIT cell levels were significantly higher in synovial fluid than in peripheral blood. Our study primarily demonstrates that MAIT cells are numerically and functionally deficient in SLE. In addition, we report a novel finding that this MAIT cell deficiency is associated with NKT cell deficiency and elevated PD-1 expression. These abnormalities possibly contribute to dysregulated mucosal immunity in SLE.

Immunological in vivo effects of B7-H1 deficiency

B7-H1 regulatory protein, a member of the B7-H family, plays a crucial role in the modulation of immune response in healthy steady-state conditions as well as in different pathologies. B7-H1 knockout mice represent an important model to elucidate further molecular and cellular mechanisms involved, among others, in autoimmunity development and cancer progression. However, a deep immunologic characterization of this model is not complete yet. This study examined the role of B7-H1 in vivo further by direct comparison of specifically phenotyped spleen immune-cell subpopulations and their activation and naïve/memory state as well as cytokine profile in wild-type and B7-H1 knockout mice. Our results demonstrated that B7-H1 deficiency in vivo modulates several immunological parameters, including the amount and composition of Gr1+CD11b+ myeloid population, the composition and activation state of the DC compartment, the frequency and status of NK and NKT cells, B-cells, naïve/memory state of CD8 T-cells and production of IL-2 and IL-10 cytokines. Moreover, we observed an increase in the PD-1 expression in the immune cells in B7-H1 knockout mice compared to the wild-type animals. Valuing the importance of B7-H1 knockout mice for their use in disease models, these data underline the role of B7-H1 in vivo also in healthy state and should be taken into account in future studies on this immunosuppressive molecule.

English

Infection is one of the most common causes of death in β-thalassemia patients. This may be due in part to an underlying immunological abnormality. During the past decade, a subset of CD3+ T cells that express both CD4+CD8+ (DP) T-cells were discovered and have been described in several pathological conditions. However, phenotypic characterization of this unique T-lymphocyte subset in patients with β-thalassemia has not yet been investigated. Flow cytometry was used to determine the frequency of such CD4+CD8+(DP) cells in concert with frequencies of CD4+, CD8+, NKT cells and γδ-TCR T-lymphocytes in the peripheral blood of β-thalassemia/HbE patients. The frequencies of these lymphocyte subsets were compared with those in blood samples from healthy volunteers. The results showed that the frequency of lymphocytes was significantly increased in splenectomized β-thalassemia/HbE patients but the frequencies of CD3+, CD4+ and CD8+ T-lymphocytes were not significantly different among the studied groups. However, analysis of unconventional T-lymphocytes revealed a significant increase in the frequency of CD4-CD8- in splenectomized β-thalassemia/HbE patients. The frequencies of CD4-CD8dim and CD4+CD8+ in β-thalassemia/HbE patients were similar to the controls. Further classification of the CD4+CD8+ cells revealed that β-thalassemia/HbE patient expressed significantly high levels of CD4brightCD8dim, with a marked increase found in non-splenectomized patients. Furthermore, significant increases in the frequency of γδ-TCR and NKT cells were also demonstrated in these splenectomized β-thalassemia/HbE patients. Our findings show the alteration of unconventional T-lymphocyte subsets in β-thalassemia/HbE patients, which may be responsible or may reflect the impaired immune response in β-thalssemia disease.

Programmed death-1 receptor suppresses γ-IFN producing NKT cells in human tuberculosis

IFN-γ biased Th1 effector immune response is crucial for containment of Mycobacterium tuberculosis infection. Various T cell subsets with regulatory function dictate the generation of Th1 like cells. NKT cells are a specialized T cell subset known to be activated early in immune response and control T cell response via release of immunoregulatory cytokines like IFN-γ, IL-4 and IL-10. M.tuberculosis, with abundance of its cell wall lipids may potently activate NKT cells resulting in cytokine production and PD-1 expression. In this study, among 49 treatment naive active pulmonary tuberculosis patients, we found a higher percentage of PD1(+) NKT cells correlating with sputum bacillary load. Furthermore, blocking PD-1 increased the number of IFN-γ producing NKT cells by inhibiting their apoptosis. Moreover, peripheral frequency of NKT cells declined with therapy suggesting their role in host T cell response. In this study, we concluded that PD-1 preferentially induces apoptosis of IFN-γ producing NKT cells while sparing NKT cells that produce IL-4. Such a polarized NKT cell function may impose a Th2 bias on the ensuing effector T cell response leading to inefficient clearance of M.tuberculosis. Inhibiting PD-1 may therefore alter the T cell response in favor of the host by rescuing type 1 NKT cells from apoptosis and boosting Th1 effector T cell functions against M.tuberculosis.

Lymphocytes Degranulation in Liver in Hepatitis C Virus Carriers Is Associated With IFNL4 Polymorphisms and ALT Levels

The polymorphisms of IFNL4 are strongly associated with both spontaneous hepatitis C virus (HCV) clearance and response to peg-IFN-α/ribavirin treatment. To further establish the biological effects of the IFNL4 and rs1297860 variations, we studied the activity of liver immune cells. Fresh liver samples were collected from HCV-infected patients before any treatment and from NASH patients as controls. Degranulation activity of each lymphocyte type was assessed by the surface expression of CD107a. IFNL4 polymorphisms and HCV genotypes were determined. In the liver, frequency of CD107a(+) immune cells was significantly higher in HCV patients compared to NASH patients. Higher degranulation activity was observed in lymphocytes of HCV patients with favorable IFNL4 genotypes compared to patients with unfavorable genotypes. Multivariate regression analyses indicated that serum ALT levels were dependent on both Metavir activity score and frequency of CD107a positive NKT cells. The high level of degranulation activity observed before treatment was associated with a high HCV RNA decline at the early stage of peg-IFN-α/ribavirin treatment in patients with favorable genotypes. These data underline that intrahepatic lymphocyte degranulation activity in HCV-infected patients is associated with IFNL4 polymorphisms and contributes to the clearance of HCV in patients with favorable genotypes under antiviral therapy.

Polymorphisms in the CD1d promoter that regulate CD1d gene expression are associated with impaired NKT cell development.

CD1d-restricted NKT cells comprise an innate-like T cell population that exerts significant influence over early events in the developing immune response. The frequency of NKT cells is highly variable in humans and in mice, but the basis for this variability remains unclear. In this study, we report a striking deficiency of type I NKT cells in the wild-derived inbred strains PWD/PhJ, SPRET/EiJ, and CAST/EiJ. Investigation of the underlying basis for the lack of type I NKT cells revealed that one strain, PWD/PhJ, exhibited a significant impairment in thymocyte and splenocyte CD1d gene and protein expression. Accordingly, both thymocytes and bone marrow-derived dendritic cells from PWD mice exhibited a significant impairment in the ability to present α-galactosylceramide to NKT cells. The impaired PWD CD1d gene expression was due to impaired CD1d promoter activity. Fine-mapping of the promoter activity revealed that two single nucleotide substitutions at positions -331 and -164 in the proximal promoter were each sufficient to account for the diminished PWD CD1d promoter activity. Examination of the strain distribution pattern of these polymorphisms revealed that, of 19 strains analyzed, only PWD and PWK mice possessed both CD1d promoter polymorphisms. A subsequent examination of the PWK strain revealed that it also exhibited impaired thymocyte CD1d expression and very low numbers of NKT cells. Taken together, these results provide new insight into the control of CD1d gene expression, and they have implications for the evolution of CD1d and type I NKT cells.

Rôle régulateur des lymphocytes NKT dans la prévention du diabète de type 1

Type 1 diabetes is an autoimmune disease resulting from the destruction of pancreatic β cells by the immune system. NKT cells are innate-like T cells that can exert potent immuno-regulatory functions. The regulatory role of NKT cells was initially proposed after the observed decreased frequency of this subset in mouse models of type 1 diabetes, as well as in patients developing various autoimmune pathologies. Increasing NKT cell frequency and function prevent the development of type 1 diabetes in mouse models. Several mechanisms including IL-4 and IL-10 production by NKT cells and the accumulation of tolerogenic dendritic cells are critical for the dampening of pathogenic anti-islet T cell responses by NKT cells. Importantly, these cells can at the same time prevent diabetes and promote efficient immune responses against infectious agents. These results strengthen the potential role of NKT cells as a key target for the development of therapeutic strategies against type 1 diabetes.

Role of ovarian hormones in T-cell homeostasis: From the thymus to the periphery

The study explored the putative role of ovarian hormones in the peripubertal remodelling of peripheral T-cell compartment. Ovariectomy at age of 1 month enhanced the peripubertal rise in CD4+ and CD8+ cell numbers in peripheral blood (PB) and spleen from 2-month-old rats. This reflected maintenance of thymopoietic efficiency at the prepubertal level (judging by numbers of the most mature CD4+ and CD8+ thymocytes and recent thymic emigrants) and alterations in T-cell survival/proliferation in the periphery. Compared with age-matched controls, the frequency of apoptotic cells among CD8+ peripheral blood lymphocytes (PBLs) and CD4+ and CD8+ splenocytes was diminished in ovariectomized (Ox) rats, at least partly, due to lower CD95 surface density. The diminished frequency of the apoptotic T splenocytes could also be associated with the rise in the amount of splenic IL-7 mRNA. Additionally, the latter finding was consistent with the augmented proliferation of CD4+ and CD8+ splenocytes. However, the enhanced proliferation of these cells could also be linked to the rise in IL-2 receptor surface density. This increase was related to the enhanced splenic TNF-α mRNA expression. Additionally, ovariectomy led to the phenotypic alterations in the major PBL and splenic T-cell subsets by diminishing/preventing the peripubertal changes in the frequency of cells at distinct stages of post-thymic differentiation/maturation (recent thymic emigrants, mature naïve and memory cells), and by decreasing the frequency of NKT cells within peripheral CD8+ subsets. In addition to numerical and phenotypic changes in T-cell compartment (due to the lack of ovarian hormone action at both the thymic and peripheral level), Ox rats exhibited a much larger delayed-type hypersensitivity (DTH) response compared with age-matched controls. This suggested the augmented T-cell-mediated immune response in Ox rats compared with aged-matched controls.

Greater Expansion of IFN-<i>γ</i><sup>﹣</sup> CD4<sup>+</sup> NKT Cells in HIV-1 Compared with HIV-2-Infected Subjects with Preserved CD4<sup>+</sup> T Cell Counts

Context: Human Natural Killer T cells are T lymphocytes that express an invariant αβ T cells receptors and NK cells receptors. They regulate innate and adaptive immune response but are susceptible to HIV-1 infection. Objective: We compare the frequency and the activity of NKT cells in HIV-1 and HIV-2 infected individuals with CD4+ counts greater than 500/mm3 using flow cytometry after overnight stimulation with phytohemagglutinin (PHA). Results: The frequency of NKT cells was similar between both groups and also to sero-negative control subjects. There were also no significant differences in the proportions of total NKT cells and the CD4+ NKT subset that secreted interferon gamma (IFN-γ) after polyclonal stimulation. However, there was a significantly higher frequency of IFN-γ﹣ CD4+ NKT cells in HIV-1-infected compared with HIV-2 infected subjects (p = 0.043). Conclusion: These data suggest there is no relationship between the functional activity of NKT cell subsets and the total NKT cell population in HIV infection. The expansion of IFN-γ﹣ CD4+ NKT cells in HIV-1 infection may serve as target for viral infection and may eventually result in their depletion during chronic infection.

The Interaction between IL-18 and IL-18 Receptor Limits the Magnitude of Protective Immunity and Enhances Pathogenic Responses following Infection with Intracellular Bacteria

The binding of IL-18 to IL-18Rα induces both proinflammatory and protective functions during infection, depending on the context in which it occurs. IL-18 is highly expressed in the liver of wild-type (WT) C57BL/6 mice following lethal infection with highly virulent Ixodes ovatus ehrlichia (IOE), an obligate intracellular bacterium that causes acute fatal toxic shock-like syndrome. In this study, we found that IOE infection of IL-18Rα(-/-) mice resulted in significantly less host cell apoptosis, decreased hepatic leukocyte recruitment, enhanced bacterial clearance, and prolonged survival compared with infected WT mice, suggesting a pathogenic role for IL-18/IL-18Rα in Ehrlichia-induced toxic shock. Although lack of IL-18R decreased the magnitude of IFN-γ producing type-1 immune response, enhanced resistance of IL-18Rα(-/-) mice against Ehrlichia correlated with increased proinflammatory cytokines at sites of infection, decreased systemic IL-10 production, increased frequency of protective NKT cells producing TNF-α and IFN-γ, and decreased frequency of pathogenic TNF-α-producing CD8(+) T cells. Adoptive transfer of immune WT CD8(+) T cells increased bacterial burden in IL-18Rα(-/-) mice following IOE infection. Furthermore, rIL-18 treatment of WT mice infected with mildly virulent Ehrlichia muris impaired bacterial clearance and enhanced liver injury. Finally, lack of IL-18R signal reduced dendritic cell maturation and their TNF-α production, suggesting that IL-18 might promote the adaptive pathogenic immune responses against Ehrlichia by influencing T cell priming functions of dendritic cells. Together, these results suggested that the presence or absence of IL-18R signals governs the pathogenic versus protective immunity in a model of Ehrlichia-induced immunopathology.

The changes and the significance of cellular immune function in peripheral blood of patients with multidrug-resistant and extensively drug-resistant pulmonary tuberculosis

To explore the expressions and the significance of CD(3)(+)CD(16)(+)CD(56)(+) NKT cells, CD(3)(-)CD(16)(+)CD(56)(+) NK cells and T lymphocyte subsets in peripheral blood of patients with multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) pulmonary tuberculosis. The data of 316 patients with pulmonary tuberculosis hospitalized in Shanghai Pulmonary Hospital from January 2008 to June 2009 were retrospectively analyzed, of whom 119 were newly diagnosed, and 197 were retreated patients. There were 204 males and 112 females, aged from 17 - 88 years, mean (44 ± 16) years. According to the results of drug-resistance, these patients were divided into a MDR group, an XDR group and a sensitive group. There were 146 patients in the MDR group, with 102 males and 44 females, aged from 19 - 84 years, mean (42 ± 16) years. There were 77 patients in the XDR group, with 42 males and 35 females, aged from 18 - 88 years, mean (50 ± 16) years. There were 93 patients in the susceptible group, with 60 males and 33 females, aged from 17 - 83 years, mean (43 ± 19) years. According to the distribution of cavitation in lung fields, these patients were also divided into 1 - 2 lung field affected group (n = 132), 3 - 4 lung field affected group (n = 49) and 5 - 6 lung field affected group (n = 9). The frequencies of NKT cells, NK cells and T cells from whole blood were tested by flow cytometry. Rank test (SAS software) was used for statistic analyses. The expression rate of NKT cells and NK cells was the highest in the XDR group [11% (6% - 16%) and 7% (4% - 12%)], as compared to the MDR group [8% (5% - 14%) and 6% (4% - 11%)], and the susceptible group [7% (4% - 11%) and 5% (3% - 9%)], the difference being statistically significant (H = 6.478 - 8.369, P < 0.05). The expression rate of the NKT (14 ± 9)% and NK cells (11 ± 7)% in males of the XDR group was significantly higher than that in females [NKT (9 ± 5)% and NK cell (6 ± 4)%], while CD(4) (38 ± 10)% and CD(4)/CD(8) (1.9 ± 1.3) were significantly lower than those of the females [CD(4) (44 ± 10)% and CD(4)/CD(8)(2.2 ± 0.7)], the difference being statistically significant (z = -2.91 - -2.79, P < 0.05, P < 0.01). The expression rate of CD(4) (42 ± 9)% was the highest, but CD(8) (22 ± 8)% was the lowest in the 1 - 2 lung field group. While in the 5 - 6 lung field group, the expression rate of CD(4) (36 ± 11)% was the lowest, CD(8) (28 ± 12)% was the highest, and CD(4)/CD(8) (1.5 ± 0.8) was the lowest, the difference being statistically significant (H = 8.404 - 16.175, P < 0.01). With the increasing level of drug resistance, the expression rate of NKT cells and NK cells increased, while the expression of T cell subsets did not change. The value of CD(4) and CD(4)/CD(8) in peripheral blood decreased, but CD(8) increased as the extent of cavitation increased in these patients. The impairment of cellular immune function in XDR-TB was more prominent in male patients.

Dynamic changes and clinical significance of HBcAg18-27 specific cytotoxic T lymphocytes in acute hepatitis B patients

This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.

High Frequency and Cell Dose of Invariant NKT Cells In the Graft Are Associated with Lack of Clinically Significant Acute Gvhd In T Cell-Replete Sibling Allografts

AbstractAbstract 2539Invariant NKT (iNKT) cells are a potent, CD1d-restricted immunomodulatory subset of T cells that regulate a variety of experimental immune responses including alloreactivity and acute graft versus host disease (aGVHD).However, their role in human immune responses and in particular in the regulation of clinical aGVHD remains unknown. Given that the frequency of iNKT cells in the blood of normal individuals varies up to 1000-fold we surmised that there was likely to be similar variability in their frequency in peripheral blood stem cell (PBSC) grafts and that the graft iNKT cell dose might be important in the development of aGVHD. To address these hypotheses, using multiparameter flow-cytometry, we determined in G-CSF mobilised PBSC grafts of 61 sibling donors the frequency and the cell dose (in × 106 cells/Kg) of CD3+TCRVbeta11+Valpha24+ iNKT cells as well as of effectors (i.e., CD3+ T cells and CD3-CD56+CD16+ NK cells) or regulators (CD3+CD161+ T cells and CD3+CD4+CD25++FoxP3+ Tregs) of alloreactivity and aGVHD.The median (minimum-maximum) frequency of T cells in the grafts was 62.3% (44.8%–80.7%) of the mononuclear cells and the dose of T cells given with the graft 179.8 (28.7–607.3), while for the NK cells it was 3.3% (0.15%–19.7%) and 9 (0.27–99.6) respectively. The frequency of the CD3+CD161+ cells was 10.5% of the total T cells (1.2%–32.5%) and the dose 17.3 (1.3–120.2). The frequency of Tregs was 5.9% of the CD3+CD4+ population (1.7%–11.9%) and the dose given 6.1 (1.1–36.4). Unlike these cell populations and similar to peripheral blood, iNKT cell content in the grafts varied up to 1000-fold with median frequency 0.045% of the total T cells (0.001%–1.07%) and cell dose 0.075 (0.0014–1.6). There was no correlation between donor age or sex with the frequency or cell dose of any the above cell populations.To assess the role of graft iNKT cells in the development of aGVHD, we selected 41 of the 61 cases, whose G-CSF mobilised PBSC graft was used for a T cell replete, sibling HLA-identical allograft (myeloablative n=34, RIC n=7) for the treatment of haematological malignancies: CML (n=16), AML (n=16), ALL (n=4), MDS (n=3) and myeloma (n=2). aGvHD prophylaxis was cyclosporine-A plus methotrexate (n=39) or cyclosporine-A only (n=2). Only patients who survived >100 days were included unless aGVHD developed earlier.Twenty one of the 41 recipients (51%) developed overall grade 0-I aGVHD (group 1) and 20 (49%) grade II-IV aGVHD (group 2). We found no difference in the CD34+ cell dose (median 5.14 vs 5.95, Mann Whitney p = 0.48), sex mismatched allografts (6 cases in each aGVHD group, chi square = 0.92), donor age (42.3yrs vs 46.1yrs, p = 0.4) or recipient age (44.2yrs vs 48yrs, p = 0.4) between the two groups. Similarly, there was no difference in the frequency or cell dose of CD3+ cells (63.3% vs 62.3% p = 0.9 and 179.8 vs 176.5 p = 0.9), CD3+CD161+ cells (14.1% vs 9%, p = 0.17 and 25.8 vs 19.6 p = 0.42), NK cells (3.5% vs 2.9% p = 0.36 and 10.8 vs 9.9 p = 0.35) and Tregs (6.3% vs 5.7% p = 0.68 and 6.1 vs 5.9 p = 0.9). In contrast, there was a significant difference in the graft iNKT cell content between the two aGVHD groups. Specifically, group 1 (grade 0-I) compared to group 2 (grade II-IV) received grafts with an almost 3-fold higher frequency (0.067% vs 0.024% p = 0.026) and dose (0.181 vs 0.07, p = 0.027) of iNKT cells. Further analysis of the relative role of the CD4+ and CD4- iNKT cell subsets revealed that the latter accounted for the protective effect of the high graft iNKT cell content (frequency 0.05% vs 0.014% p = 0.016 and cell dose 0.072 vs 0.023 p = 0.01) but not their CD4+ counterpart (0.038% vs 0.011% p = 0.12 and 0.051 vs 0.029 p = 0.14).Thus, this study is the first to demonstrate that graft iNKT cells are an important determinant of aGVHD in humans and suggests that enrichment of the graft with iNKT cells might be a useful strategy to prevent clinically significant aGVHD. Disclosures:No relevant conflicts of interest to declare.

Innate Immune Function in Placenta and Cord Blood of Hepatitis C – Seropositive Mother-Infant Dyads

Vertical transmission accounts for the majority of pediatric cases of hepatitis C viral (HCV) infection. In contrast to the adult population who develop persistent viremia in ∼80% of cases following exposure, the rate of mother-to-child transmission (2–6%) is strikingly low. Protection from vertical transmission likely requires the coordination of multiple components of the immune system. Placenta and decidua provide a direct connection between mother and infant. We hypothesized that innate immune responses would differ across the three compartments (decidua, placenta and cord blood) and that hepatitis C exposure would modify innate immunity in these tissues. The study was comprised of HCV-infected and healthy control mother and infant pairs from whom cord blood, placenta and decidua were collected with isolation of mononuclear cells. Multiparameter flow cytometry was performed to assess the phenotype, intracellular cytokine production and cytotoxicity of the cells. In keeping with a model where the maternal-fetal interface provides antiviral protection, we found a gradient in proportional frequencies of NKT and γδ-T cells being higher in placenta than cord blood. Cytotoxicity of NK and NKT cells was enhanced in placenta and placental NKT cytotoxicity was further increased by HCV infection. HCV exposure had multiple effects on innate cells including a decrease in activation markers (CD69, TRAIL and NKp44) on NK cells and a decrease in plasmacytoid dendritic cells in both placenta and cord blood of exposed infants. In summary, the placenta represents an active innate immunological organ that provides antiviral protection against HCV transmission in the majority of cases; the increased incidence in preterm labor previously described in HCV-seropositive mothers may be related to enhanced cytotoxicity of NKT cells.

Early Changes in Frequency of Peripheral Blood Lymphocyte Subpopulations in Severe Traumatic Brain‐Injured Patients

Infections are leading causes of increased morbidity and mortality of severe traumatic brain-injured (STBI) patients. The mechanism underlying the susceptibility to the infections is still unexplained. The purpose of the study was to investigate changes in frequency of leucocytes subpopulations in peripheral blood of patients with STBI during the course of intensive care treatment. Twenty patients with STBI were included in the study. Healthy age- and sex- volunteers served as control. Peripheral blood samples were taken from these patients at day 1, 4 and 7, and peripheral blood mononuclear cells (PBMC) were isolated. The percentage of T, B lymphocyte, NK and NKT cells as well as monocytes was analysed by simultaneous detection of surface antigens using fluorochrome-conjugated monoclonal antibodies. The two major subsets of T lymphocytes (CD3(+)CD56(-)CD4(+) and CD3(+)CD56(-)CD8(+)) and NK cells (CD3(-)CD56(+dim) and CD3(-)CD56(+bright)) were also analysed by flow cytometry. Extracranial infections were presented in 55% patients with STBI. At day 4, the percentage of T lymphocytes with cytotoxic phenotype significantly diminished and their numbers restored at day 7. The frequency of NKT cells showed the identical time-dependent pattern, whereas the percentage of NK cells diminished on day 4 but did not restore after 7 days. The frequency of B lymphocytes did not change significantly during the time investigated, whereas the percentage of monocytes increased immediately after the injury and gradually diminished. The decrease in cells with cytotoxic phenotype might explain high incidence of susceptibility to infection of patients with STBI.

Testing the NKT cell hypothesis in lenalidomide-treated myelodysplastic syndrome patients

Myelodysplastic syndrome (MDS) comprises a group of clonal bone marrow disorders characterized by ineffective hematopoiesis and increased predisposition to acute myeloid leukemia. The causes of MDS remain poorly defined, but several studies have reported the NKT cell compartment of patients with MDS is deficient in number and functionally defective. In support of a central role for NKT cells, a pilot clinical study reported that lenalidomide (an approved treatment for MDS) increased NKT cell numbers in patients with MDS, and several in vitro studies showed lenalidomide specifically promoted NKT cell proliferation and cytokine production. We tested this in a much larger study and confirm a moderate in vitro augmentation of some NKT cell functions by lenalidomide, but find no impact on the NKT cell compartment of patients treated with lenalidomide, despite a consistently positive clinical response. We further show that the frequency and cytokine production of NKT cells is normal in patients with MDS before treatment and remains stable throughout 10 months of lenalidomide therapy. Collectively, our data challenge the concept that NKT cell defects contribute to the development of MDS, and show that a clinical response to lenalidomide is not dependent on modulation of NKT cell frequency or function.

Circulating subsets and CD4+CD25+ regulatory T cell function in chronic inflammatory demyelinating polyradiculoneuropathy

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an inflammatory disease of the peripheral nervous system that is probably autoimmune in origin. Different components of the adaptive and innate immunity may be responsible for the aberrant response towards nerve antigens. To investigate this, we examined lymphocyte subsets and regulatory T cell (Treg) function in the blood of CIDP patients, healthy controls (HC) and subjects with non-immune mediated neuropathies (other neuropathies, ON). We used flow cytometry to determine the frequency of monocytes, B cells, natural killer (NK) and NK-T cells, total and activated CD4+ and CD8+ T cells, effector memory and central memory CD4+ and CD8+ T cells, and CD4+CD25highFoxp3+ Tregs. Treg function was studied after polyclonal stimulation and antigen specific stimulation with myelin protein peptides in CIDP and HC. There was an increased frequency of monocytes (p = 0.02) and decreased frequency of NK cells (p = 0.02) in CIDP compared with HC but not ON. There were no significant differences in other populations. Treg function was impaired in CIDP compared to HC (p = 0.02), whilst T cell proliferation to myelin protein peptides before and after depletion of Tregs was not different between patients and controls. This study shows increased circulating monocytes and reduced NK cells in CIDP. Although Treg frequency was not altered, we confirm that Tregs display a defect of suppressive function. Myelin protein peptides were not the target of the altered peripheral regulation of the immune response. The mechanisms of peripheral immune tolerance in CIDP and their relevance to the pathogenesis deserve further exploration.

Widespread natural variation in murine natural killer T‐cell number and function

Natural killer T (NKT) cells comprise a novel T-lymphocyte subset that can influence a wide variety of immune responses through their ability to secrete large amounts of a variety of cytokines. Although variation in NKT-cell number and function has been extensively studied in autoimmune disease-prone mice, in which it has been linked to disease susceptibility, relatively little is known of the natural variation of NKT-cell number and function among normal inbred mouse strains. Here, we demonstrate strain-dependent variation in the susceptibility of C57BL/6J and BALB/cJ mice to NKT-mediated airway hyperreactivity, which correlated with significant increases in serum interleukin-4 (IL-4) and IL-13 elicited by the synthetic glycosphingolipid alpha-galactosylceramide. Examination of NKT-cell function revealed a significantly greater frequency of cytokine-producing NKT cells in C57BL/6J versus BALB/cJ mice as well as significant differences in the kinetics of NKT-cell cytokine production. Extension of this analysis to a panel of inbred mouse strains indicated that variability in NKT-cell cytokine production was widespread. Similarly, an examination of NKT-cell frequency revealed a significantly greater number of liver NKT cells in the C57BL/6J mice versus BALB/cJ mouse livers. Again, examination of a panel of inbred mouse strains revealed that liver NKT-cell numbers were quite variable, spanning over a 100-fold range. Taken together, these results demonstrate the presence of widespread natural variation in NKT-cell number and function among common inbred mouse strains, which may have implications for the examination of the influence of NKT cells in immune responses and disease pathogenesis among different genetic backgrounds.

NKT cell development in the absence of the autoimmune regulator gene (Aire)

Autoimmune regulator gene (Aire)-deficient mice develop an array of autoimmune lesions that reflect failures of immune tolerance. Negative selection is clearly compromised in these mice, but there is evidence to suggest that other mechanisms of tolerance might also be affected, including a possible impairment of regulatory T cell (Treg) development. Studies to date have failed to demonstrate any significant impact on the development or function of the FOXP3+ Treg compartment, but NKT cells represent a distinct regulatory cell lineage that also develop in the thymus and which are known to influence self-tolerance. Aire-related defects coincide with NKT cell deficiencies in a number of animal models, but the direct consequence of Aire-deficiency on NKT cell development has not been established. In this study, we demonstrate that the frequency, distribution and cytokine production of NKT cells and their subsets is principally normal in Aire-deficient mice. We conclude that Aire has little or no effect on regulatory T cell development in general and NKT cells in particular.

Apolipoprotein E-Mediated Immune Regulation in Sepsis

Lipids and lipoproteins have emerged as key constituents of the immune response to microbial infection. We, therefore, sought to understand the complex interaction between lipoprotein metabolism and sepsis. Apolipoprotein E (apoE), a component of plasma lipoproteins, has been suggested to bind and traffic Ags for NKT cell activation. However, apoE's role in sepsis has not been demonstrated. In this study, we examined the effect of exogenous apoE in a rat model of septic peritonitis, induced by cecal ligation and puncture. We demonstrate that 48 h after serial injections of apoE, septic mortality increased in a dose-dependent manner. While sepsis resulted in increased splenic and decreased hepatic and circulating NKT cell populations, serial injections of apoE for 24 h after cecal ligation and puncture increased the frequency, cell number, and BrdU uptake in splenic and hepatic NKT cell populations, while concomitantly depleting these populations in the circulation. These changes were correlated with elevated alanine amino transferase levels, an indicator of liver injury. Interestingly, while sepsis increased hepatic T cell apoptosis and necrosis, apoE reversed these changes. apoE also promoted increases in predominantly Th1 cytokine levels in sera and a decrease in IL-4, the main NKT cell-derived Th2 cytokine. Consequently, apoE treatment is associated with increased sepsis-induced mortality, and increased NKT cell frequency and proliferation in the liver and spleen, with concomitant decreases in these NKT cell parameters in the peripheral circulation. apoE treatment also promoted a Th1 cytokine response, increased the degree of liver injury, and decreased apoptosis in hepatic lymphocytes.