The Expression And Functional Role Of ILT2on Nk Cell In Patients With Tuberculosis

Tuberculosis (TB) is the second leading cause of death from an infectious diseaseworldwide. The incident of TB increases due to human immunodeficiency virus (HIV)infection, movement of population, drug-resistant bacteria and use ofimmunosuppressive agent. In2012, an estimated8.6million people developed TB and1.3million died from the disease. Globally an estimated450,000people developedMDR-TB. There are an estimated5million TB patients in China, and0.13milliondeaths every year. Despite high rate of Mycobacterium tuberculosis infection inhumans, only5-10%of infected people develop into active TB in their life time. Thesudies show that the occurrence, development and outcome of TB do not only dependon the number of Mycobacterium tuberculosis and virulence, but largely on immuneresponses of the host.Host innate immune defense provides early protection against M. tuberculosisinfection and also plays an important role in initiating acquired immunity. K cells,macrophages, monocyte and neutrophilic leukocytes are the main cells involved in theinnate immunity and they can play activities mainly through discharging perforin andgranular enzyme, expression of ligand and secretion of interferon without the help ofantigen sensitization in advance. NK cells belong to innate immunity and they havecytotoxic and cytokine-producing activities and have immunoregulatory properties inthe same time. It has been proved that NK cell functions are regulated by differentactivating and inhibitory surface receptors, in order to maintain a balanced immuneresponse toward infection. Ig-like transcript2(ILT2)(also called CD85j, LILRB1,LIR-1) is a type I transmembrane protein and contains four immunoreceptortyrosine-based inhibition motifs (ITIMs) in its cytoplasmic domain.The receptor is involved in negative signaling through recruiting Src homology2(SH2)domain-containing proteins, such as SHP-1, upon tyrosine phosphorylation of ITIMs.ILT2is expressed on NK cells, T lymphocytes, B lymphocytes, monocytes anddendritic cells, and it interacts with MHC class I molecules including HLA-A,-B,-C,-E and-G and some viral proteins such as UL18protein coded by HCMV. Thefunctional role of ILT2in anti-TB immunity is not clear. I n this study, we investigatedthe function of ILT2on NK cells during TB infection, including detecting theexpression of ILT2on NK cell subsets in peripheral blood from patients with TB,relationship between the expression of ILT2and the severity of pulmonary TB,correlation of expression of ILT2with cytolytic activity and cytokine secretionfunction of NK cells in peripheral blood from patients with active in order to determinewhether ILT2has any influence on NK cell function and the role of ILT2on aspontaneous apoptosis of CD56dimNK cells during active TB infection. Our studydemonstrated that patients with active pulmonary TB had significantly higherfrequency of ILT2-expressing CD56dimCD16+NK cells than tuberculin-positivehealthy controls, which correlated with disease severity of pulmonary TB.ILT2-expressing CD56dimNK cells had reduced expression of CD107a and IFN-, andblockage of ILT2signaling could rescue functional defect of CD56dimCD16+NK cells,our result illustrated that ILT2might promote apoptosis of CD56dimNK cells duringactive TB infection. The results suggested an inhibitory role of ILT2on CD56dimCD16+NK cells during active TB infection in humans, which provided a deeper insight ofimmunity mechanism of TB and provide basis for establishment of treatment plan ofanti-TB by strengthening the function of NK cells. Therefore, the research on theexpression and functional role of ILT2on NK cell in patients with tuberculosis wascarried out.In the first part, we studied the frequency of NK cells and frequency of ILT2expressing NK cells in peripheral blood of active pulmonary TB and analyzed therelationship beween frequencyof ILT2-expressing CD56dimCD16+NK cells and diseaseseverity of pulmonary TB. The study recruited seventy patients with active pulmonary TB and67purified protein derivation (PPD)-positive healthy group.The pulmonary TBgroup were further divided into two groups: smear/culture-positive group (n=41) andsmear/culture-positive group (n=29).Firstly, anticoagulant fresh blood were collected, PBMCs were purified by densitygradient centrifugation.The cells were stained with FITC-, PE-Cy5-or PE-Cy7-labeledanti-human CD3, CD56and CD16monoclonal antibodies. The frequency of NK cellswere determined with flow cytometry. The results showed that patients with active TBhad significantly lower frequencies of CD3-CD56+NK cells (2.3%;1.2%-4.0%,p<0.0001) than PPD-positive healthy controls (7.8%;6.5%-11.9%) and withsignificantly lower frequencies of CD3-CD56dim(3.6%;2.2-6.7%, p<0.0001) andCD3-CD56brightNK cells (0.3%;0.2-0.6%, p=0.0002) than PPD-positive healthycontrols (9.2%;6.2-14.1%and0.5%;0.3-0.6%). The results suggested that patientswith active TB had significantly lower frequencies of NK cells and subtypes.Secondly, anticoagulant fresh blood were collected, PBMCs were were purifiedby density gradient centrifugation. The cells were stained with FITC-, PE-, PE-Cy5-orPE-Cy7-labeled anti-human CD3, ILT2, CD56and CD16monoclonal antibodies.Frequency of ILT2expressing NK cells in peripheral blood of pulmonary TB wereanalyzed with flow cytometry. The results showed that the frequency ofCD56dimCD16+NK cells that expressed ILT2was45.7%(33.9-56.2%) in patients withactive pulmonary TB and19.1%(9.9-30.0%) in tuberculin-positive healthy controls(p<0.0001). In contrast, the frequency of ILT2-expressing CD56brightCD16+/-NK cellsbetween patients with active pulmonary TB (2.6%;1.4-4.9%) and tuberculin-positivehealthy controls (2.4%;0.7-5.7%) was similar (p=0.5163). The results suggested thatpatients with active TB had increased proportion of ILT2-expressing CD56dimCD16+NK cells.Lastly, the frequency of ILT2-expressing CD56dimCD16+NK cells was comparedbetween M. tuberculosis smear/culture-positive and-negative patients. The results showed thatpatients with M. tuberculosis smear/culture-positive result had significantly higher frequency of ILT2-expressing CD56dimCD16+NK cells (55.3%;45.4-59.9%) thanthose with M. tuberculosis smear/culture-negative result (32.9%;23.3-42.0%),p<0.0001. The result suggested that ILT2expression on CD56dimCD16+NK cellscorrelated with disease severity of pulmonary TB.In the second part, the relationship between ILT2and CD107a expression on NKcells and the expression of IFN-γ in NK cells were carried out to ascertain thefunctions of ILT2on NK cells during TB infection. The percentage of apoptotic cellsin ILT2+CD56dimNK cells were compared with that in ILT2-CD56dimNK cells toascertain the functions of ILT2on the apoptosis of CD56dimNK cells during active TBinfection. The study recruited38patients with active pulmonary TB, with M.-tuberculosis smear/culture-positive result.Firstly, PBMCs of15patients with active pulmonary TB was incubated with targetcells K562at the ratio of5:1and anti-human CD107a antibody was added, CD107aexpressing on ILT2+CD56dimNK cells and ILT2-CD56dimNK cells were determined byflow cytometry. The results showed that ILT2+CD56dimNK cells had significantlylower CD107a expression than ILT2-cells when cells were incubated with target cellline K562(p=0.0169). The result suggested that ILT2-expressing CD56dimNK cellshad functional defect of cytotoxic activity.Secondly, PBMCs of10patients with active pulmonary TB were incubatedtogether with K562cells and monensin was added after incubation. Cells were firststained with fluorescence labelled monoclonal antibodies, and then were fixed andpermeabilized. The expression of IFN-γ in NK cells was detected by FITC-labeledanti-IFN-γ monoclonal antibody and analyzed by flow cytometer. The results showedthat ILT2+CD56dimNK cells had significantly reduced IFN-production comparedwith ILT2-CD56dimNK cells (p=0.0038). The result suggested that ILT2-expressingCD56dimNK cells had functional defect of IFN-secretion.Thirdly, PBMCs were cultured with blocking anti-ILT2antibody or isotype controlantibody prior to incubation with the target cell line K562. CD107a expression on NK cells was measured. The results showed that locking of ILT2signaling resulted inincreased expression of CD107a on CD56dimCD16+NK cells (p=0.0223). The resultsuggested that blockade of ILT2signaling can rescue functional defect ofCD56dimCD16+NK cells.Lastly, Spontaneous apoptosis of NK cells were determined by staining withAnnexin V and PI antibody and analyzed by flow cytometry. The percentage ofapoptotic cells in ILT2+CD56dimNK cells were compared with that in ILT2-CD56dimNK cells. The percentage of apoptotic cells was significantly higher in ILT2+CD56dimNK cells than in ILT2-CD56dimNK cells (p=0.0335). The result suggested that ILT2might promote apoptosis of CD56dimNK cells during active TB infection.On the basis of the strips mengtioned above, the expression and functional role ofILT2on NK cell in patients with tuberculosis was carried out with flow cytometry,andthe results illustrated that patients with active TB had decreased proportion of NK cellsand subtypes, patients with active TB had increased proportion of ILT2-expressingCD56dimCD16+NK cells, and the frequency of ILT2-expressing CD56dimCD16+NKcells correlated with disease severity of pulmonary TB, ILT2can inhibit the functionof CD56dimNK cells of cytolytic activity and cytokines secretion, the result alsosuggested that ILT2might promote apoptosis of CD56dimNK cells during active TBinfection.In short, The results suggested an inhibitoryrole of ILT2on CD56dimCD16+NKcells during active TB infection in humans, which provided a deeper insight ofimmunity mechanism of TB and provide basis for establishment of treatment plan ofanti-TB by strengthening the function of NK cells.