Latently infected cells are highly heterogeneous and very scarce. Even the most sensitive assays detect only several hundred infected cells in a typical peripheral blood sample. It is therefore both desirable and potentially feasible to analyse this small cell population extensively, but first, the cells must be identified by screening millions of lymphocytes.
Flow cytometry can achieve adequate throughput, but there is no published method to identify infected cells. One assay in development detects induced intracellular expression of p24 antigen [24
]. As for any single-cell analysis, the limit of detection is constrained by the number of cells that can be practically analysed. The high throughput of modern flow cytometers makes it feasible to analyse tens of millions of cells, and thus to detect induced p24 expression at levels below one per million lymphocytes.
Many alternative approaches to high-throughput single-cell analysis are now available. Among these, CyTOF and microengraving have already been used to characterize primary lymphocytes in unprecedented detail and are particularly promising for the near future. CyTOF couples flow cytometry, which analyses single cells, with time-of-flight (TOF) mass spectrometry, which analyses single molecules [11
]. In this method, detection antibodies are conjugated to stable isotopes of transition metals, instead of fluorescent labels. This allows multiplexed detection and quantification of over 30 different molecules without the complexity of fluorescence compensation needed for multiparameter flow cytometry [25
CyTOF has been used to characterize leukocyte phenotype in terms of over 30 different cellular proteins measured simultaneously [12■■
]. Although CyTOF requires cell destruction for analysis, it has been used to characterize cell function by simultaneously measuring phosphorylated and unphosphorylated forms of key proteins in signal transduction pathways [26
]. This analysis of intracellular phosphorylation was sufficient to classify cells, that is to predict the cell surface phenotype. Whether similar changes distinguish latently infected cells is not yet clear, but CyTOF provides a method to identify any such signatures.
Microengraving involves simultaneous culture of single lymphocytes in an array of subnanolitre wells [27
]. Secreted molecules are captured on an antibody-functionalized surface, which can be periodically replaced to measure secretion kinetics [29
]. Medium can also be changed, allowing the responses of individual lymphocytes to different stimuli to be monitored for up to 2 weeks. After analysis of the capture surface, cells of special interest can be selectively recovered from an array [30
]. This technology has recently been used for high-throughput quantitative analysis of individual primary T lymphocytes by measuring cytokine production kinetics [13
], and by relating cytokine production to the cytolytic activity of both CD8+
] and natural killer (NK) cells [32
]. Nucleic acids can be amplified and detected by performing single-cell ‘digital’ PCR within the culture array. Microengraving has been used to clonally expand HIV-specific CD8+
T lymphocytes with high throughput [16■■
], but it might instead be used to characterize cellular reservoirs of HIV infection. Other microfluidic culture systems have the potential to provide similar information [33
], but none of these has yet achieved the practicality and experimental throughput demonstrated in these recent microengraving studies.
Microengraving and CyTOF offer the obvious prospect of characterizing the diverse latently infected cell population, but it is also noteworthy that single-cell analysis inherently provides quantitative measurements of cell populations. Regardless of the parameters measured, any method that identifies latently infected cells individually would also provide a count of their number. These techniques should therefore measure the size of the latent reservoir with the same precision and accuracy as ddPCR, while providing enormous additional detail about the heterogeneity of this population. If the response of latently infected cells to proposed adjunctive therapies is as heterogeneous as the cellular phenotypes suggest, this additional information may prove essential for monitoring and evaluation of eradication studies.