Key Points• The frequency of CD161 ϩϩ MAIT cells is dramatically decreased in the blood of HIVinfected patients, and they are nonrecoverable with HAART.• Gut sequestration and apoptosis in response to bacterial signals may, amongst others, be mechanisms that contribute to this. IntroductionThe natural course of human immunodeficiency virus type 1 (HIV-1) infection is associated with progressive immune dysfunction, perturbation of immune-cell subsets and increased opportunistic infections. In early disease, there is a dramatic loss of CD4 ϩ T cells from the gastrointestinal tract resulting in impaired mucosal immunity, reduced peripheral CD4 ϩ T-cell count, and increased systemic T-cell activation. [1][2][3][4] These factors contribute to an increased susceptibility to infection with specific organisms such as Mycobacterium tuberculosis and Candida albicans. [5][6][7] In addition, more recent evidence suggests an important role for the loss of CD8 ϩ T cells in susceptibility to bacterial pneumonia and all-cause mortality in HIV infection. 8 MAIT cells are a distinct subset of tissue-infiltrating lymphocytes with antibacterial functions that account for up to one-third of the CD8 ϩ T-cell population in the blood of healthy individuals. [9][10][11] MAIT cells are identified by expression of a semi-invariant T-cell receptor (TCR), iV␣7.2, 10,12,13 which recognizes ligands presented by MHC class I related (MR1) protein. 14 MR1 presentation occurs on dendritic cells, monocytes, and lung epithelial cells in response to bacterial pathogens. 9,10,12 MAIT cells are activated in vitro in an MR1-dependent fashion by a range of bacterial and fungal pathogens, including Escherichia coli, M tuberculosis, and C albicans, 9,10 and in mouse models have been shown to provide protection against bacterial infection. 10,15 In addition, MAIT cells have been shown to be lost from the blood and present in the lungs of patients with active tuberculosis, suggesting they may play an important role in host immunity to M tuberculosis. 9,10 Specific subsets of CD4 ϩ and CD8 ϩ T cells, termed Th17 and Tc17, are defined by their ability to produce IL17A and are important in the regulation of mucosal integrity and antibacterial immunity. [16][17][18][19][20] Early in HIV infection, Th17 cells are lost from the gastrointestinal tract, but may be restored through long-term highly active antiretroviral therapy (HAART) concurrent with a reduction in immune activation levels. 21 The loss of this IL17A and Submitted June 12, 2012; accepted November 26, 2012. Prepublished online as Blood First Edition paper, December 18, 2012; DOI 10.1182 DOI 10. /blood-2012 *C.C. and J.E.U. contributed equally to this work.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 7, 2018. by guest www.bloodjournal.org...

HighlightsThe requirement for protocol adaptation from qPCR to ddPCR is characterised.A potential loss of sensitivity for ddPCR at low target numbers is reported.A new bioinformatic tool ‘definetherain’ to improve droplet calling at low input target numbers is devised and applied.‘Definetherain’ is made available free of charge and open access at www.definetherain.org.uk.

Macrophage infection is considered to play an important role in HIV-