Alterations in circulating lymphoid cell populations in systemic small vessel vasculitis are non-specific manifestations of renal injury.

Innate lymphocyte populations, such as innate lymphoid cells (ILCs), γδ T cells, invariant natural killer T (iNK T) cells and mucosal-associated invariant T (MAIT) cells are emerging as important effectors of innate immunity and are involved in various inflammatory and autoimmune diseases. The aim of this study was to assess the frequencies and absolute numbers of innate lymphocytes as well as conventional lymphocytes and monocytes in peripheral blood from a cohort of anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV) patients. Thirty-eight AAV patients and 24 healthy and disease controls were included in the study. Patients with AAV were sampled both with and without immunosuppressive treatment, and in the setting of both active disease and remission. The frequencies of MAIT and ILC2 cells were significantly lower in patients with AAV and in the disease control group compared to healthy controls. These reductions in the AAV patients remained during remission. B cell count and frequencies were significantly lower in AAV in remission compared to patients with active disease and disease controls. Despite the strong T helper type 2 (Th) preponderance of eosinophilic granulomatosis with polyangiitis, we did not observe increased ILC2 frequency in this cohort of patients. The frequencies of other cell types were similar in all groups studied. Reductions in circulating ILC2 and MAIT cells reported previously in patients with AAV are not specific for AAV, but are more likely to be due to non-specific manifestations of renal impairment and chronic illness. Reduction in B cell numbers in AAV patients experiencing remission is probably therapy-related.

Circulating CD56dim natural killer cells and CD56+ T cells that produce interferon-γ or interleukin-10 are expanded in asymptomatic, E antigen-negative patients with persistent hepatitis B virus infection.

Infection with hepatitis B virus (HBV) can result in spontaneous resolution or chronic infection, which can remain asymptomatic or can progress to cirrhosis and/or hepatocellular carcinoma. The host immune response is thought to be a major determinant of the outcome of HBV infection and virus-specific cytotoxic T lymphocytes (CTL) can mediate immunity against the virus and cause liver pathology. Antigen-nonspecific innate lymphocytes may also contribute to HBV infection and liver disease, therefore, we examined the frequencies, phenotypes, cytolytic activities and cytokine profiles of circulating natural killer (NK) cells, CD1d-restricted invariant natural killer T (iNKT) cells and CD56(+) T cells in 102 asymptomatic HBV-infected patients and compared them with those in 66 uninfected control subjects. NK cells expressing low levels of CD56 (CD56(dim)) and CD56(+) T cells were significantly expanded in the circulation of HBV-infected patients compared with control subjects. CD1d expression and iNKT cell frequencies were similar in both groups. Despite these expansions, we did not detect augmented natural or cytokine-induced cytotoxicity in the HBV-infected subjects. All lymphocyte populations studied produced interferon-γ (IFN-γ) significantly more frequently when taken from HBV-infected patients compared with when taken from healthy controls. Additionally, NK cells from the patients more frequently produced interleukin-10. As our HBV-infected cohort consisted of asymptomatic patients with low viral loads, we propose that CD56(dim) NK cells and CD56(+) T cells control HBV infection by noncytolytic mechanisms.

Glucagon-like peptide-1 (GLP-1) and the regulation of human invariant natural killer T cells: lessons from obesity, diabetes and psoriasis.

AIMS/HYPOTHESIS: The innate immune cells, invariant natural killer T cells (iNKT cells), are implicated in the pathogenesis of psoriasis, an inflammatory condition associated with obesity and other metabolic diseases, such as diabetes and dyslipidaemia. We observed an improvement in psoriasis severity in a patient within days of starting treatment with an incretin-mimetic, glucagon-like peptide-1 (GLP-1) receptor agonist. This was independent of change in glycaemic control. We proposed that this unexpected clinical outcome resulted from a direct effect of GLP-1 on iNKT cells. METHODS: We measured circulating and psoriatic plaque iNKT cell numbers in two patients with type 2 diabetes and psoriasis before and after commencing GLP-1 analogue therapy. In addition, we investigated the in vitro effects of GLP-1 on iNKT cells and looked for a functional GLP-1 receptor on these cells. RESULTS: The Psoriasis Area and Severity Index improved in both patients following 6 weeks of GLP-1 analogue therapy. This was associated with an alteration in iNKT cell number, with an increased number in the circulation and a decreased number in psoriatic plaques. The GLP-1 receptor was expressed on iNKT cells, and GLP-1 induced a dose-dependent inhibition of iNKT cell cytokine secretion, but not cytolytic degranulation in vitro. CONCLUSIONS/INTERPRETATION: The clinical effect observed and the direct interaction between GLP-1 and the immune system raise the possibility of therapeutic applications for GLP-1 in inflammatory conditions such as psoriasis.

Altered natural killer cell subset distributions in resolved and persistent hepatitis C virus infection following single source exposure.

BACKGROUND: Natural killer (NK) cells may be impaired in patients with persistent hepatitis C virus (HCV) infection, but studies to date have yielded inconsistent findings due to patient and virus heterogeneity and difficulties obtaining appropriate controls. AIMS: To overcome these variables, we have examined numbers, phenotypes, cytotoxic activities and cytokine profiles of circulating NK cells from Irish women who acquired infection through administration of HCV genotype 1b-contaminated anti-D immunoglobulin from a single source and matched controls. RESULTS: Comparing 29 women who developed persistent infection with 21 who spontaneously resolved infection and 26 controls, we found that NK cell numbers were consistently lower in the persistently infected group (p = 0.02 and 0.002). This decrease was due to depletions of NK cells expressing low levels of CD56 (CD56(dim) NK cells; p = 0.004 and 0.0001), whilst CD56(bright) NK cells were expanded (p = 0.004 and 0.0001). Compared to HCV resolvers, CD56(dim) NK cells from persistently infected patients less frequently expressed CD16 and more frequently expressed NKG2A/C/E. These phenotypic changes did not significantly affect natural or interleukin-2-induced cytotoxicity by peripheral blood mononuclear cells against K562 and Daudi targets. Greater frequencies of CD56(bright) NK cells from chronic HCV patients produced interferon-gamma compared with HCV responders (p = 0.05) and controls (p = 0.0001) after phorbol ester stimulation in vitro. CONCLUSIONS: Alterations in NK subset distributions in chronic HCV infection may explain why previous reports of impaired NK cell functions were difficult to confirm. Altered NK cell functions may contribute to impaired cellular immune responses and chronicity of disease following HCV infection.

Genetic association of the HLA DRB1 gene locus on chromosome 6p21.3 with schizophrenia.

OBJECTIVE: The authors investigated the human leukocyte antigen (HLA) DRB1*04 gene in schizophrenic patients because it is positively associated with rheumatoid arthritis, an autoimmune disease that exhibits a strong negative association with schizophrenia. The HLA DQB1*0602 allele was also studied because of previous reports of genetic association between it and schizophrenia. Maternal HLA was investigated because of the reported association between prenatal influenza and schizophrenia and the central role of HLA molecules in the immune response to viral infections. METHOD: Polymerase chain reactions and sequence-specific oligonucleotide probes were used to genotype 94 unrelated patients with DSM-III-R schizophrenia, 92 mothers of schizophrenic offspring who were not related either to each other or to the 94 patients, and 177 healthy comparison subjects. RESULTS: The frequency of DRB1*04 alleles was significantly lower in both the schizophrenic patients and the unrelated mothers of schizophrenic offspring than in the healthy comparison subjects. No significant differences were found for DQB1*0602. CONCLUSIONS: DRB1*04 alleles may partially account for the genetic predisposition to schizophrenia. The association reported here may be explained by genetic linkage or by an autoimmune pathophysiology for a proportion of schizophrenia cases. Alternatively, it may be that maternal B lymphocytes that do not express the DR4 antigen encoded by DRB1*04 respond to influenza virus by producing antibodies that perturb neurodevelopment, thus underpinning a proportion of schizophrenia cases.

Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization.

Murine and human studies have demonstrated that the normal liver contains significant numbers of resident lymphocytes that have functions distinct from those found in blood and other organs. To characterize these cells requires the isolation of viable lymphocytes that can be analysed by flow cytometry and in functional assays. The techniques classically used to isolate single cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue. The aim of this study was to determine the effect of these procedures on surface molecule expression and lymphocyte function and to optimise an isolation technique that minimises these effects. Mechanical homogenisation of liver tissue alone resulted in low viable lymphocyte yields but these were improved by the combined use of mechanical and enzymatic techniques. A mean yield of 2.3 x 10(6) lymphocytes with a mean viability was 88.8% was obtained from 200 mg wedge biopsy samples of normal adult human liver using a combination of gentle mechanical dissociation followed by digestion with collagenase type IV and DNase I. These cells were suitable for phenotypic characterisation by flow cytometry. They also retained their ability to grow in vitro, to respond to cytokines and activation stimuli, to mediate cytotoxic killing of target cells, and to produce inflammatory and regulatory cytokines.

HLA genotyping of colorectal carcinoma in the Chinese population.

The HLA-DR genotypes of 61 primary colorectal carcinomas obtained from patients of Chinese origin were determined by using DNA-RFLP. No increase or decrease of a particular HLA genotype could be ascertained with the disease, although we detected an antigen frequency of 29.5% for the serologically ill-defined DR"X3" specificity. We identified and sequenced HLA-DRB1 and DRB3 genes from the DR"X3" haplotype. The DR"X3" DRB1 gene was found to be identical to DRB1*1201 (DR5[w12]). A unique observation is its unusual linkage with DRB3*0101 (DRw52a) or DRB3*0301 (DRw52c) instead of the usual linkage with DRB3*0201/2 (DRw52b). These associations are rare in whites and blacks.

HLA DQA, DQB, and DRB genotyping by oligonucleotide analysis: distribution of alleles and haplotypes in British caucasoids.

A precise method for comprehensive HLA DQA and DQB genotyping using gene amplification and hybridization with sequence-specific oligonucleotide (SSO) probes is described. Twenty-four SSO probes were used to detect all DQ allotypes defined by nucleotide sequence variation in the second exons of the DQ genes, using a standard set of conditions for all probes at each locus. Five hundred individuals were genotyped for 8 DQA1 and 16 DQB1 alleles by using this method and for 33 alleles of the DRB1, DRB3, DRB4, and DRB5 genes by using previously described SSO probes. The 4-locus DQB1-DQA1-DRB1-DRB3/4/5 haplotypes present were characterized on the basis of known linkage disequilibrium between class II alleles. Fifty-two different haplotypes that have previously been described were further characterized at the nucleotide sequence level and two novel haplotypes were identified. The distributions of these alleles and haplotypes in 177 randomly selected healthy Caucasoid controls from the United Kingdom are reported. These results identify further haplotypic diversity in the HLA class II region, even though strong linkage disequilibrium exists between the DR and DQ gene loci.

Human small intestinal epithelial cells secrete interleukin-7 and differentially express two different interleukin-7 mRNA Transcripts: implications for extrathymic T-cell differentiation.

The small intestinal epithelium, composed of epithelial cells (EC) and intraepithelial T lymphocytes, is exposed to numerous ingested antigens. Small intestinal EC may act as accessory and/or antigen presenting cells for intestinal T cells, some of which may mature extrathymically and regulate local immunity and tolerance. Since interleukin-7 (IL-7) plays an essential role in T cell maturation and activation, we examined its expression by human small intestinal EC. IL-7 was detected by ELISA in supernatants from 4 of 4 epithelial layer (EpL) cultures. Using RT-PCR, IL-7 mRNA was detected in 4 EpL studied, and two distinct IL-7 transcripts were identified in 3 of the 4. The ratios of the intensities of the larger to the smaller bands varied amongst individuals. Furthermore, the intensity ratios were higher in whole-thickness intestine and lamina propria preparations than in their corresponding EpL. This is the first report of the expression of two IL-7 transcripts in human intestine and of IL-7 secretion by human small intestinal EpL cells. This supports the hypothesis that small intestinal EC may influence differentiation and/or activation of neighboring T cells. The differential expression of the two transcripts may have important implications for immune regulation in the intestinal epithelium.

Natural T cells in the human liver: cytotoxic lymphocytes with dual T cell and natural killer cell phenotype and function are phenotypically heterogenous and include Valpha24-JalphaQ and gammadelta T cell receptor bearing cells.

The adult liver contains lymphocytes with a unique phenotypic distribution compared to blood and other organs. We have characterized a human lymphocyte population that exhibits dual T cell and natural killer (NK) cell phenotype and function, denoted natural T (NT) cells, in nine normal adult liver specimens. Flow cytometry revealed that up to 55% (mean 27%) of hepatic (but <6% of peripheral) CD3+ lymphocytes expressed CD56, CD161 and/or one or more of the killer inhibitory receptors (KIR) p58.1, p58.2, p70 and CD94. NK function was attributed to the CD3+CD56+ cells by the demonstration that hepatic, but not peripheral, CD3+ lymphocytes could be induced to lyse NK-sensitive K562 target cells, while CD56- cells from both compartments could not. Three color flow cytometric analysis of fresh hepatic cells indicated that CD3+CD56+ NT cells can be either CD8+, CD4+ or CD4 CD8-, they express alphabeta or gammadelta T cell receptors (TCR) and CD161 and KIRs, but rarely CD16. Hepatic NT cells predominantly express the mature/activated CD45RO and CD56dim phenotypes. Analysis of mRNA production by isolated NT cells indicated a preferential usage of the invariant CD1-restricted Valpha24-JalphaQ TCR. The presence of such large numbers of chronically activated NT cells provides compelling evidence that the liver has unique immunoregulatory functions.

Polymorphism in the human complement C4 genes and genetic susceptibility to autoimmune hepatitis.

Susceptibility to autoimmune hepatitis is associated with the HLA-DR3 and DR4 haplotypes, but which genes are directly involved in the pathogenesis, has not been established. Low levels of complement component C4 and elevated frequencies of C4 null allotypes have been described in patients, suggesting that the C4 genes, which are closely linked with the HLA loci, may play a role. We therefore examined restriction fragment length polymorphisms in the C4 and 21-hydroxylase genes, and determined HLA-A and B phenotypes, and HLA-DR, DQ and DP genotypes in a large series of Caucasoid patients with autoimmune hepatitis and matched controls. A DNA deletion of the C4A gene and the 21-hydroxylase A pseudogene was found to be present in 50% of patients compared to 23% of controls (Pc < 0.005, relative risk = 3.3). This increase, however, appears to be due to linkage disequilibrium with HLA-DR52a which was most strongly associated with the disease. Complete C4A deficiency, determined by homozygosity for the deletion increased the risk to 18.1 (16% versus 1%, Pc < 0.005), suggesting an additional role for C4 in disease susceptibility. C4 deletions were associated with an increased mortality and tendency to relapse whilst on treatment but did not correlate with age of onset of disease. Our data suggest that MHC-encoded susceptibility to autoimmune hepatitis is polygenic, involving the HLA-DR genes plus other loci, and C4 deficiency may be a marker of disease susceptibility and/or severity.

Characterization of NKR+ T-cell subsets in human bone marrow: implications for immunosurveillance of neoplasia.

In addition to hematopoietic progenitors, human bone marrow contains mature T/NK lymphocytes. Valpha24Vbeta11 NKT-cells, a subset of NK receptor+ (NKR+) T-cells in humans, are rare in bone marrow, suggesting the presence of other NKR+ T-cells which may contribute to tumor surveillance. NKR+/- T-cells were examined in blood (PB), and bone marrow from donors (DM) and patients with active hematopoietic malignancy (PM), or in remission (PR). T-cells in PR & PM were enriched for CD56+ and CD57+ subsets, compared to DM. All marrow NKR+/- T-cell subsets were more activated than PB. PM and, surprisingly, PR marrow contained more activated cells than DM. CD8+ cells were significantly increased in all patient marrows and there was evidence of the formation of an effector/memory pool in malignant marrow. These data suggest that NKR+ T-cell enrichment in human bone marrow that has been exposed to neoplastic transformation is compatible with a role in localized tumor surveillance/eradication.

Butylated hydroxyanisole and lung tumor development in A/J mice.

A diet containing 0.75% butylated hydroxyanisole (BHA) did not enhance the development of lung tumors in A/J mice if fed for 8 weeks after administration of urethan, benzo[a]pyrene (B[a]P), or dimethylnitrosamine (DMN). Prefeeding animals with BHA partially protected animals against the tumorigenic effect of urethan and B[a]P. Partial protection was also seen in animals given B[a]P and then exposed to BHA in the diet. The two isomers of BHA (3-tert.-butyl-4-hydroxyanisole and 2-tert.-butyl-4-hydroxyanisole) were synthesized and injected ip. They failed to enhance lung tumor development. It is concluded that BHA is not a promoting agent as is butylated hydroxytoluene (BHT) for lung tumors in mice. One possible explanation is that BHA in the diet does not produce the extensive cell proliferation seen in the lungs of mice fed BHT.

HLA-DRB and DQB typing by a combination of serology, restriction fragment length polymorphism analysis and oligonucleotide probing.

DNA sequencing has revealed extensive polymorphism within the HLA-DRB and DQB genes of the major histocompatibility complex. At least 43 alleles of the DRB loci and 13 alleles of the DQB1 locus are currently recognized. Identification of these alleles can be performed phenotypically by cellular and serological techniques or genotypically by restriction fragment length polymorphism (RFLP) analysis and sequence-specific oligonucleotide (SSO) probing of DNA amplified by the polymerase chain reaction (PCR). However, each method has its technical limitations so the tissue typing laboratory must therefore choose from the range of available techniques to maximize the efficiency and accuracy of HLA typing. In this paper we describe a scheme for the accurate determination of all the serologically defined DRB and DQB allotypes using a combination of serology, RFLP analysis and PCR-SSO probing. The efficiencies of serology versus RFLP analysis for the DR typing of 800 individuals, and RFLP analysis versus PCR-SSO probing for the DQB typing of 317 individuals, are compared and the merits of each technique discussed. This scheme, which types for both HLA-DRB and DQB with an accuracy approaching 100%, is now routinely employed in all HLA studies of disease and transplantation in our laboratory.