CD8+/CD161++ mucosal-associated invariant T-cell levels in the colon are restored on lsong-term antiretroviral therapy and correlate with CD8+ T-cell immune activation
Greathead, Louisea; Metcalf, Rebeccaa; Gazzard, Brianb; Gotch, Francesa; Steel, Alanc; Kelleher, Petera,b
aImmunology Section, Division of Infectious Diseases, Imperial College Chelsea & Westminster Hospital
bHIV & Sexual Health Directorate
cDepartment of Gastroenterology, Chelsea and Westminster Hospital NHS Foundation Trust, London, UK.
Correspondence to Peter Kelleher, Immunology Section, Imperial College, Chelsea & Westminster Hospital, 369 Fulham Road, SW10 9NH London, UK. E-mail: firstname.lastname@example.org
Received 10 September, 2013
Revised 15 May, 2014
Accepted 15 May, 2014
Mucosal-associated invariant T (MAIT) cells are tissue-homing T cells recently implicated in HIV pathogenesis. We found that the proportion of MAIT cell in blood and colon of HIV+ patients are reduced in untreated infection. Antiretroviral therapy restored colonic but not blood MAIT cell percentages. We observed a negative correlation between colonic MAIT cells and T-cell activation in blood and suggest mucosal MAIT cell depletion may contribute to systemic immune activation in HIV infection.
Mucosal-associated invariant T (MAIT) cells are a recently described innate-like mucosal-homing T cell subset which express the semi-invariant T cell receptor Vα7.2, interleukin-18R and are strongly positive for CD161 . MAIT cells play a key role in host immunity against bacteria and fungi by recognition of vitamin B metabolites presented by the MHC class 1b-related protein MR1 . Two recent studies have shown that antiretroviral therapy (ART) fails to restore MAIT cell percentages in the blood [3,4]. Damage to the mucosal epithelium resulting in microbial translocation has been proposed as a mechanism for the immune activation seen in HIV+ patients . We hypothesized that depletion of MAIT cells in the colon of HIV+ patients would correlate with increased immune activation and markers of microbial translocation.
We analysed paired blood and colonic samples from 37 HIV-1-infected patients (12 with detectable viraemia and 25 on suppressive ART) and 22 HIV-uninfected controls. The control patients were undergoing colonoscopy at Chelsea & Westminster NHS Trust due to a personal or family history of large bowel polyps or cancer.
Biopsies were collected 25–30 cm from the anal verge of the sigmoid colon using Jumbo EndoJaw 3.7 mm forceps (Olympus) and processed as previously described to obtain mucosal mononuclear cells (MMCs) . Peripheral blood mononuclear cells and MMCs were stained with Violet Dead Cell Stain (Invitrogen) and the following antibodies: CD16-FITC, CD3-V500, CD8-AF700, CD19-V450, CD14-V450, CD161-PE-CY5, HLADR-FITC, CD38-PE (all BD Bioscience); CD45RO-PE-TR (Beckman Coulter); CD27-APC-Efluor780 (eBioscience); and CD4-QDot605 (Invitrogen). The cells were acquired on an LSRII (BD Bioscience) and analysed in FlowJo v8.8.6 (TreeStar). After gating to exclude doublets, aggregates, and dead cells, MAIT cells were identified as CD3+CD4−CD8+ lymphocytes with bright expression of CD161. To exclude NKT cells, we confirmed that the population was CD16-negative. T-cell activation was identified on memory CD8+ cells by bright CD38 expression.
Quantitative real-time PCR was performed using Platinum Syber Green Supermix (Invitrogen) in an Applied Biosystems ABI PRISM 7000 with a standard curve made from purified Escherichia coli (Affymetrix). Primers used were: forward (5′-3′-TGG AGC ATG TGG TTT AAT TCG A), reverse (5′-3′-TGC GGG ACT TAA CCC AAC A) (Eurofins MWG Operon), and PCR conditions were: 10 min 95oC, 40× 40 s 94oC, 45 s 60oC, 45 s 72oC. A PCR run was accepted if the slope of the standard fell between −3.6 and −3.3, and the curve fit was greater than 0.985. The samples were run in duplicate and repeated if the SD of the quantification cycle (Cq) was greater than 0.500. Soluble CD14 (R&D Systems) was measured in plasma according to the manufacturers’ instructions. Kruskal–Wallis test with Dunns multiple correction was used to compare groups and correlations were performed using Spearman's rho in Prism 4 (GraphPad).
We saw a significant reduction in the proportion of blood CD8+ T cells that express CD161++ in ART-naive and ART-treated HIV patients compared to healthy controls (Table 1; P < 0.001). The expression of CD161++ on CD8+ T cells isolated from colon samples was also significantly reduced in viraemic HIV (P = 0.005), but was similar to controls in patients receiving ART. We found no correlation between MAIT cell proportions in the blood compared to the colon in our control or HIV groups. We also found no correlation between mucosal MAIT cells and the time since diagnosis in ART-naive patients group or time on treatment in the ART-treated cohort.
CD8+ T-cell activation was significantly increased in both the HIV groups (Table 1; P < 0.001) compared to controls, and levels of activation were significantly higher in viraemic compared to aviraemic HIV (P < 0.001). We found a significant inverse correlation between mucosal MAIT cell proportions and levels of CD8+ T-cell activation in both the HIV and control groups (HIV Spearman's rho = −0.434, P = 0.008; controls Spearman's rho = −0.512, P = 0.018). We also found significantly raised levels of sCD14 in the plasma of both HIV groups compared to controls (P = 0.006), but this did not correlate with MAIT cell levels. There was a trend for increased bacterial 16s rDNA in viraemic patients (Table 1; P = 0.051), but we found no correlations with blood or colonic MAIT cells.
Our data confirm results of recent studies showing a significant reduction in the proportion of blood CD161++CD8+ T cells in viraemic and ART-treated HIV patients compared to healthy controls [3,4]. However, in contrast to the study by Leeansyahet al., we found a significant depletion of MAIT cells isolated from colon biopsies in viraemic HIV patients compared to controls. Differences in sampling sites, markers used to identify MAIT cells and patient characteristics may in part account for discrepancy between the two studies. It has been proposed that the rate of loss of MAIT cells in mucosal tissues is much slower than that seen in blood and the failure of ART to restore this lymphocyte subset in blood argues for treatment of HIV as early as possible to preserve host immunity against bacterial and fungal infections . Our results show that the kinetics of MAIT cell loss in untreated HIV infection appears to be similar in the blood and mucosa, and we demonstrate that ART appears to be effective in restoring the MAIT cell population. Although we did find a negative correlation between T-cell activation and mucosal MAIT cell proportions in both our HIV and control populations, we did not find any associations with evidence of microbial translocation. However, microbial translocation is more frequently seen in patients with advanced chronic disease  and our cohort included patients with more recent infection. It is possible that the relationship between colonic MAIT cell proportions and microbial translocation may be more apparent if one were to evaluate bacterial translocation directly at mucosal sites .
In conclusion, we show that, although MAIT cells remain diminished in blood even during ART, colonic MAIT cell populations restore to normal levels and negatively correlate with CD8+ T-cell immune activation in HIV infection. We suggest that this correlation with immune activation, identified as a better marker of HIV disease progression than CD4+ cell counts and viral loads , implies an important regulatory function for MAIT cells located in the gastrointestinal tract.
We would like to thank all the patients who generously participated in this study and we acknowledge the support provided by the Endoscopy Unit at the Chelsea &Westminster NHS Foundation Trust.
L.G. performed all the flow cytometry, ELISA and the 16s rDNA experiments. R.M. established the 16s rDNA assay and supervised the molecular microbial translocation assays. A.S. recruited patients for paired blood and colonic samples and supervised the endoscopic investigations. P.K., A.S., and B.G. designed the study. L.G., F.G., A.S., B.G., and P.K. wrote the article.
Financial support was provided by St Stephens AIDS Trust and the Westminster Medical School Research Trust.
Conflicts of interest
There are no conflicts of interest.
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© 2014 Lippincott Williams & Wilkins, Inc.
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