The HLA region in ANCA-associated vasculitis: characterisation of genetic associations in a Scandinavian patient population.

OBJECTIVE: The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are inflammatory disorders with ANCA autoantibodies recognising either proteinase 3 (PR3-AAV) or myeloperoxidase (MPO-AAV). PR3-AAV and MPO-AAV have been associated with distinct loci in the human leucocyte antigen (HLA) region. While the association between MPO-AAV and HLA has been well characterised in East Asian populations where MPO-AAV is more common, studies in populations of European descent are limited. The aim of this study was to thoroughly characterise associations to the HLA region in Scandinavian patients with PR3-AAV as well as MPO-AAV. METHODS: Genotypes of single-nucleotide polymorphisms (SNPs) located in the HLA region were extracted from a targeted exome-sequencing dataset comprising Scandinavian AAV cases and controls. Classical HLA alleles were called using xHLA. After quality control, association analyses were performed of a joint SNP/classical HLA allele dataset for cases with PR3-AAV (n=411) and MPO-AAV (n=162) versus controls (n=1595). Disease-associated genetic variants were analysed for association with organ involvement, age at diagnosis and relapse, respectively. RESULTS: PR3-AAV was significantly associated with both HLA-DPB1*04:01 and rs1042335 at the HLA-DPB1 locus, also after stepwise conditional analysis. MPO-AAV was significantly associated with HLA-DRB1*04:04. Neither carriage of HLA-DPB1*04:01 alleles in PR3-AAV nor of HLA-DRB1*04:04 alleles in MPO-AAV were associated with organ involvement, age at diagnosis or relapse. CONCLUSIONS: The association to the HLA region was distinct in Scandinavian cases with MPO-AAV compared with cases of East Asian descent. In PR3-AAV, the two separate signals of association to the HLD-DPB1 region mediate potentially different functional effects.

A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin-3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

Relation between HLA and copy number variation of steroid 21-hydroxylase in a Swedish cohort of patients with autoimmune Addison's disease.

OBJECTIVE: Autoantibodies against the adrenal enzyme 21-hydroxylase is a hallmark manifestation in autoimmune Addison's disease (AAD). Steroid 21-hydroxylase is encoded by CYP21A2, which is located in the human leucocyte antigen (HLA) region together with the highly similar pseudogene CYP21A1P. A high level of copy number variation is seen for the 2 genes, and therefore, we asked whether genetic variation of the CYP21 genes is associated with AAD. DESIGN: Case-control study on patients with AAD and healthy controls. METHODS: Using next-generation DNA sequencing, we estimated the copy number of CYP21A2 and CYP21A1P, together with HLA alleles, in 479 Swedish patients with AAD and autoantibodies against 21-hydroxylase and in 1393 healthy controls. RESULTS: With 95% of individuals carrying 2 functional 21-hydroxylase genes, no difference in CYP21A2 copy number was found when comparing patients and controls. In contrast, we discovered a lower copy number of the pseudogene CYP21A1P among AAD patients (P = 5 × 10-44), together with associations of additional nucleotide variants, in the CYP21 region. However, the strongest association was found for HLA-DQB1*02:01 (P = 9 × 10-63), which, in combination with the DRB1*04:04-DQB1*03:02 haplotype, imposed the greatest risk of AAD. CONCLUSIONS: We identified strong associations between copy number variants in the CYP21 region and risk of AAD, although these associations most likely are due to linkage disequilibrium with disease-associated HLA class II alleles.

Strong Association of Combined Genetic Deficiencies in the Classical Complement Pathway With Risk of Systemic Lupus Erythematosus and Primary Sjögren's Syndrome.

OBJECTIVE: Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjögren's syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. METHODS: The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. RESULTS: Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 × 10-9 ) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. CONCLUSION: We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS.

Complement C4 Copy Number Variation is Linked to SSA/Ro and SSB/La Autoantibodies in Systemic Inflammatory Autoimmune Diseases.

OBJECTIVE: Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. METHODS: Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. RESULTS: A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2-33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7-5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals' capacity to deposit C4b on immune complexes. CONCLUSION: We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.

Actionable druggable genome-wide Mendelian randomization identifies repurposing opportunities for COVID-19.

Drug repurposing provides a rapid approach to meet the urgent need for therapeutics to address COVID-19. To identify therapeutic targets relevant to COVID-19, we conducted Mendelian randomization analyses, deriving genetic instruments based on transcriptomic and proteomic data for 1,263 actionable proteins that are targeted by approved drugs or in clinical phase of drug development. Using summary statistics from the Host Genetics Initiative and the Million Veteran Program, we studied 7,554 patients hospitalized with COVID-19 and >1 million controls. We found significant Mendelian randomization results for three proteins (ACE2, P = 1.6 × 10-6; IFNAR2, P = 9.8 × 10-11 and IL-10RB, P = 2.3 × 10-14) using cis-expression quantitative trait loci genetic instruments that also had strong evidence for colocalization with COVID-19 hospitalization. To disentangle the shared expression quantitative trait loci signal for IL10RB and IFNAR2, we conducted phenome-wide association scans and pathway enrichment analysis, which suggested that IFNAR2 is more likely to play a role in COVID-19 hospitalization. Our findings prioritize trials of drugs targeting IFNAR2 and ACE2 for early management of COVID-19.

Function of multiple sclerosis-protective HLA class I alleles revealed by genome-wide protein-quantitative trait loci mapping of interferon signalling.

Interferons (IFNs) are cytokines that are central to the host defence against viruses and other microorganisms. If not properly regulated, IFNs may contribute to the pathogenesis of inflammatory autoimmune, or infectious diseases. To identify genetic polymorphisms regulating the IFN system we performed an unbiased genome-wide protein-quantitative trait loci (pQTL) mapping of cell-type specific type I and type II IFN receptor levels and their responses in immune cells from 303 healthy individuals. Seven genome-wide significant (p < 5.0E-8) pQTLs were identified. Two independent SNPs that tagged the multiple sclerosis (MS)-protective HLA class I alleles A*02/A*68 and B*44, respectively, were associated with increased levels of IFNAR2 in B and T cells, with the most prominent effect in IgD-CD27+ memory B cells. The increased IFNAR2 levels in B cells were replicated in cells from an independent set of healthy individuals and in MS patients. Despite increased IFNAR2 levels, B and T cells carrying the MS-protective alleles displayed a reduced response to type I IFN stimulation. Expression and methylation-QTL analysis demonstrated increased mRNA expression of the pseudogene HLA-J in B cells carrying the MS-protective class I alleles, possibly driven via methylation-dependent transcriptional regulation. Together these data suggest that the MS-protective effects of HLA class I alleles are unrelated to their antigen-presenting function, and propose a previously unappreciated function of type I IFN signalling in B and T cells in MS immune-pathogenesis.

Immunogenetics in systemic lupus erythematosus: Transitioning from genetic associations to cellular effects.

Systemic lupus erythematosus (SLE) is a heterogeneous rheumatic autoimmune disease. Genetic studies have identified up to 100 SLE risk loci. Many of these encode proteins of importance in the immune system, but the cellular and molecular mechanisms underlying these associations are still elusive. In this review, we will highlight some of the SLE risk loci where mechanistic insights have been achieved recently by linking genetic risk polymorphisms to cellular or molecular phenotypes important for the disease process.

IL7RA genetic variants differentially affect IL-7Rα expression and alternative splicing: a role in autoimmune and infectious diseases?

Interleukin-7 receptor α chain (IL-7Rα) single nucleotide polymorphisms (SNPs) are associated with susceptibility to immunopathologies like autoimmune and inflammatory diseases. The current hypothesis about underlying mechanisms is based on the regulation of IL-7 availability for self-reactive T cells by influencing the generation of a soluble (s)IL-7Rα variant. This assumption was mainly predicated on the well-defined IL7RA SNP rs6897932, which affects alternative splicing and causes aberrant generation of the sIL-7Rα variant with potential effects on the IL-7 serum reservoir. However, more recent studies shed light on novel functions of autoimmunity risk-associated IL7RA SNPs and characterized the largely neglected effect of rs6897932 on membrane (m)IL-7Rα expression. These findings as well as a described role of impaired mIL-7Rα expression and IL7RA SNP influence on chronic infectious diseases necessitates the reevaluation of previous findings on the role of IL7RA SNPs in immunopathology.

Autoantibodies to Killer Cell Immunoglobulin-Like Receptors in Patients With Systemic Lupus Erythematosus Induce Natural Killer Cell Hyporesponsiveness.

Natural killer (NK) cell cytotoxicity toward self-cells is restrained by the inhibitory HLA class I-binding receptors CD94/NKG2A and the killer cell immunoglobulin-like receptors (KIRs). CD94/NKG2A and KIRs are also essential for NK cell education, which is a dynamic functional maturation process where a constitutive binding of inhibitory receptors to cognate HLA class I molecules is required for NK cells to maintain their full cytotoxic capacity. Previously, we described autoantibodies to CD94/NKG2A in patients with systemic lupus erythematosus (SLE). In this study we analyzed sera from 191 patients with SLE, 119 patients with primary Sjögren's syndrome (pSS), 48 patients with systemic sclerosis (SSc), and 100 healthy donors (HD) for autoantibodies to eight different KIRs. Anti-KIR autoantibodies were identified in sera from 23.0% of patients with SLE, 10.9% of patients with pSS, 12.5% of patients with SSc, and 3.0% of HD. IgG from anti-KIR-positive SLE patients reduced the degranulation and cytotoxicity of NK cells toward K562 tumor cells. The presence of anti-KIR-autoantibodies reacting with >3 KIRs was associated with an increased disease activity (p < 0.0001), elevated serum levels of IFN-α (p < 0.0001), nephritis (p = 0.001), and the presence of anti-Sm (p = 0.007), and anti-RNP (p = 0.003) autoantibodies in serum. Together these findings suggest that anti-KIR autoantibodies may contribute to the reduced function of NK cells in SLE patients, and that a defective NK cell function may be a risk factor for the development of lupus nephritis.

Two-Hit in vitro T-Cell Stimulation Detects Mycobacterium tuberculosis Infection in QuantiFERON Negative Tuberculosis Patients and Healthy Contacts From Ghana.

IFN-γ release assays [e.g., QuantiFERON (QFT)] are widely used for diagnosis of Mycobacterium tuberculosis (Mtb) infection. T-cell responses against QFT antigens ESAT6 and CFP10 are highly Mtb specific but previous studies indicated suboptimal assay sensitivity. Especially for potentially infected healthy contacts (HCs) of tuberculosis patients, alternative antigen usage and more sensitive tests may contribute to improved detection of latent Mtb infection. In a pilot case-control study of tuberculosis patients (n = 22) and HCs (n = 20) from Ghana, we performed multifaceted in vitro assays to identify optimal assay conditions. This included a two-hit stimulation assay, which is based on initial and second re-stimulation with the same antigen on d6 and intracellular IFN-γ analysis, to compare T-cell responses against ESAT6/CFP10 (E6/C10) and selected latency antigens (i.e. Rv2628, Rv1733, Rv2031, Rv3407) of Mtb. Considerable subgroups of tuberculosis patients (64%) and HCs (75%) had negative or indeterminate QFT results partially accompanied by moderate PHA induced responses and high IFN-γ background values. Intracellular IFN-γ analysis of E6/C10 specific CD4+ T-cell subpopulations and evaluation of responder frequencies had only moderate effects on assay sensitivity. However, two-hit in vitro stimulation significantly enhanced E6/C10 specific IFN-γ positive T-cell proportions especially in QFT non-responders, and in both study groups. Mtb latency antigen-specific T cells against Rv1733 and Rv2628 were especially detected in HCs after two-hit stimulation and T-cell responses against Rv2628 were highly capable to discriminate tuberculosis patients and HCs. Two-hit in vitro stimulation may improve moderate sensitivity of short term IFN-γ based assays, like QFT, to detect Mtb infection. Latency stage-specific antigens added significantly to detection of Mtb infection in HCs and tuberculosis patients with negative QFT test results.

Autoimmunity risk- and protection-associated IL7RA genetic variants differentially affect soluble and membrane IL-7Rα expression.

Interleukin-7 receptor α-chain (IL7RA) haplotypes are associated with susceptibility to autoimmune diseases including type 1 diabetes (T1D). Previous studies found lower soluble IL-7Rα (sIL-7Rα) serum levels of the protection-associated IL7RA haplotype assumed to reduce IL-7 availability for self-reactive T cells. Also, a risk-associated IL7RA haplotype is accompanied by lower sIL-7Rα serum concentrations but no underlying mechanisms have been described and the causative polymorphism remains unknown. Here, we characterized functional implications of the nonsynonymous rs1494558 (Thr66Ile), which tags the protection-associated IL7RA haplotype, in HEK293T cells and serum samples of T1D patients with different haplotype carriers. Influence of risk- and protection-associated haplotypes on IL-7Rα was analyzed. The risk-associated Ile66 variant affected gel mobility and impaired secretion of the sIL-7Rα as well as expression of the membrane-associated (m)IL-7Rα in HEK293T cells. Serum sIL-7Rα analyses confirmed differential gel mobility of the Ile66 variant and found decreased sIL-7Rα serum levels of T1D patients carrying the Ile66-tagged haplotype. Differences in glycosylation were not causative for differential mobility but enhanced the effects on impaired secretion. Comparison of protection- and risk-associated haplotypes in a cell line-based in vitro model identified dominant effects of the protective haplotype tagged by rs6897932 (Ile244) on mIL-7Rα expression, whereas the risk haplotype mainly affected the sIL-7Rα. This study identified novel functional effects of the Ile66 IL7RA variant and characterized features of autoimmunity risk- and protection-associated haplotypes. The findings add to our understanding of how these haplotypes regulate sIL-7Rα and mIL-7Rα expression in T cells causing differential susceptibility to autoimmune diseases.

An IL7RA exon 5 polymorphism is associated with impaired IL-7Rα splicing and protection against tuberculosis in Ghana.

Functional interleukin-7 receptor α-chain (IL-7Rα) genetic variants, which affect alternative splicing and expression of the soluble IL-7Rα, are associated with susceptibility to autoimmunity. We previously described aberrant IL-7Rα expression and impaired IL-7-mediated T-cell functions in tuberculosis patients. In the present study, we investigated a possible role of IL7RA gene variants. Six exonic IL7RA polymorphisms were genotyped and two minor alleles were found at lower frequencies in tuberculosis patients as compared to healthy contacts from Ghana (rs11567764, p = 0.002; rs1494558, p = 0.01). The rs11567764 polymorphism tags an IL7RA haplotype exclusively found in African populations and was predicted to affect splicing of exon 5. Reduced mRNA expression of the Δexon_5-6 variant was found in T-cells from carriers of the protective rs11567764 allele. Although we were not able to demonstrate the causative effect of rs11567764, our findings suggested functional implications of genetic variants on IL-7Rα splicing and with potential impact on T-cell protection against tuberculosis.

Interleukin-7 receptor α-chain haplotypes differentially affect soluble IL-7 receptor and IL-7 serum concentrations in children with type 1 diabetes.

BACKGROUND: Interleukin-7 receptor α-chain (IL7RA) haplotypes are associated with susceptibility for development of autoimmune diseases, including type 1 diabetes (T1D). A protective IL7RA haplotype which causes lower soluble IL-7R (sIL-7R) serum levels is hypothesized to restrict IL-7-availability for self-reactive T cells. Functional mechanisms affected by a risk-associated IL7RA haplotype are unknown. METHODS: We investigated the influence of IL7RA haplotypes (tagged by rs6897932T for the protective or by rs1494555G for the risk haplotype) on sIL-7R and IL-7 serum concentrations as well as disease manifestation of children with T1D (n = 259). Possible effects of differential IL-7 serum concentrations on IL-7-mediated in vitro T cell functions (i.e. IL-7R regulation and cytokine expression) were measured in a second study group of children with T1D (n = 42). RESULTS: We detected lower sIL-7R serum concentrations in children with T1D carrying protective or risk haplotypes as compared to reference haplotypes. sIL-7R levels were lowest in T1D children with the protective haplotype and lower IL-7 serum levels were exclusively detected in this study group. We found no evidence for dependency between IL-7 and sIL-7R serum concentrations and no association with T1D manifestation. Neither IL-7 nor sIL-7R serum levels were associated with mIL-7R regulation or IL-7-promoted T cell cytokine expression. CONCLUSIONS: Children with T1D carrying autoimmunity risk- or protection-associated IL7RA haplotypes had both lower sIL-7R serum concentrations as compared to the reference haplotype, but only T1D children with the protective haplotype had lower IL-7 serum levels. Our results suggest additional functional mechanisms of autoimmunity-associated IL7RA variants independent from sIL-7R mediated regulation of IL-7 availability for T cells.

In Vivo and Ex Vivo Inflammatory Markers of Common Metabolic Phenotypes in Humans.

BACKGROUND: Low-grade systemic inflammation (LGSI) is often characterized by elevated levels of interleukin (IL)6, tumor necrosis factor (TNF)α, and C-reactive protein (CRP). Other serum proteins, ex vivo-stimulated cytokine production, and leukocyte count have, however, also been suggested LGSI-markers, but their associations with the metabolic syndrome (MS) are less clear. We aimed to evaluate mutual relationships between in vivo and ex vivo inflammatory markers and their association with MS and its subcomponents. METHODS: A cross-sectional study of 118 overweight adults with one or several features of MS. Inflammatory markers included fasting serum levels of IL6, TNFα, CRP, and pentraxin-3 (PTX3), IL1-receptors, leukocytes, and whole-blood ex vivo-produced IL1ß, IL6, TNFα, and IL8 after lipopolysaccharide stimulation. RESULTS: All classical serum LGSI-markers correlated with each other, and IL6 and CRP were also correlated with leukocyte count. Ex vivo-produced cytokines were intercorrelated and correlated with leukocyte count, but did not correlate with the serum immune markers. MS score, body mass index, and glycated hemoglobin (HbA1c) were associated with 8%-16% higher inflammatory score per standard deviation increment (P = 0.030, 0.001, and 0.034, respectively), primarily driven by higher serum IL6. Serum PTX3 was only significantly associated with high-density lipoprotein cholesterol (1.19[1.04; 1.37], P = 0.013). HbA1c was inversely associated with surface expression of IL1R1 on monocytes and IL1R2 on granulocytes (P < 0.01) and with a 3%-9% lower ex vivo production of cytokines when adjusting for leukocyte count, as were plasma triacylglycerol (9%-10% lower IL1ß and IL6). Leukocyte count was most consistently associated with MS and its subcomponents, although not with HbA1. CONCLUSIONS: The classical fasting serum markers of LGSI and leukocyte counts associated best with measures of MS-associated LGSI, whereas ex vivo cytokine production was only associated with prevailing glycemia and dyslipidemia. Taken together, this indicates that the relationship between in vivo and ex vivo inflammatory markers is complex and may depend on the MS phenotype.

Aberrant plasma IL-7 and soluble IL-7 receptor levels indicate impaired T-cell response to IL-7 in human tuberculosis.

T-cell proliferation and generation of protective memory during chronic infections depend on Interleukin-7 (IL-7) availability and receptivity. Regulation of IL-7 receptor (IL-7R) expression and signalling are key for IL-7-modulated T-cell functions. Aberrant expression of soluble (s) and membrane-associated (m) IL-7R molecules is associated with development of autoimmunity and immune failure in acquired immune deficiency syndrome (AIDS) patients. Here we investigated the role of IL-7/IL-7R on T-cell immunity in human tuberculosis. We performed two independent case-control studies comparing tuberculosis patients and healthy contacts. This was combined with follow-up examinations for a subgroup of tuberculosis patients under therapy and recovery. Blood plasma and T cells were characterised for IL-7/sIL-7R and mIL-7R expression, respectively. IL-7-dependent T-cell functions were determined by analysing STAT5 phosphorylation, antigen-specific cytokine release and by analysing markers of T-cell exhaustion and inflammation. Tuberculosis patients had lower soluble IL-7R (p < 0.001) and higher IL-7 (p < 0.001) plasma concentrations as compared to healthy contacts. Both markers were largely independent and aberrant expression normalised during therapy and recovery. Furthermore, tuberculosis patients had lower levels of mIL-7R in T cells caused by post-transcriptional mechanisms. Functional in vitro tests indicated diminished IL-7-induced STAT5 phosphorylation and impaired IL-7-promoted cytokine release of Mycobacterium tuberculosis-specific CD4+ T cells from tuberculosis patients. Finally, we determined T-cell exhaustion markers PD-1 and SOCS3 and detected increased SOCS3 expression during therapy. Only moderate correlation of PD-1 and SOCS3 with IL-7 expression was observed. We conclude that diminished soluble IL-7R and increased IL-7 plasma concentrations, as well as decreased membrane-associated IL-7R expression in T cells, reflect impaired T-cell sensitivity to IL-7 in tuberculosis patients. These findings show similarities to pathognomonic features of impaired T-cell functions and immune failure described in AIDS patients.

Alternative Quantiferon cytokines for diagnosis of children with active tuberculosis and HIV co-infection in Ghana.

IFN-γ release assays (IGRAs) often present false-negative or indeterminate results in children with tuberculosis. HIV co-infection may contribute to decreased sensitivity of IGRAs by impairing T-cell IFN-γ expression. Measurement of alternative cytokines in QuantiFERON® (QFT) supernatants can circumvent the IFN-γ-dependency and may improve QFT sensitivity. We aimed to identify additional cytokines from QFT supernatants for detection of Mycobacterium tuberculosis infection in children with tuberculosis and HIV co-infection from Ghana. Concentrations of 18 cytokines in QFT supernatants from children (0-16 years) with tuberculosis concomitantly infected with HIV (n = 25) or without HIV (n = 24) from Ghana were measured using cytometric bead array (CBA). 29% of the children showed positive IFN-γ test results, and five cytokines, i.e., IL-6, IL-21, TNF-α, IL-1α and IP-10, detected M. tuberculosis infection with comparable or, for IL-6, with significantly higher sensitivity (59%). Increased age and HIV co-infection were associated with decreased cytokine induction, and especially IL-21 and IP-10 were less prevalent in HIV co-infected children with tuberculosis. Combined cytokine analyses increased proportions of positive tests, and a four-cytokine subset (i.e., IL-6, IL-21, IFN-γ, IL-1α) predicted 78% of the children with tuberculosis correctly. Combined evaluation of IFN-γ and alternative cytokines improved IGRA-sensitivity in children with tuberculosis.