Single-cell analysis of infected cell responses to TCR stimulation. (A) Assay method. Resting memory CD4⁺ T cells were stimulated for 24 h with bead-bound anti-CD3 and anti-CD28. IPDA analysis was then used to determine the frequency of infected cells (orange) for plating at limiting dilution. After 7–8 days, proliferation of infected cells and proviral intactness were determined by IPDA analysis of each well. Total cell proliferation in the same wells was determined by ddPCR for the host gene RPP30, and virion production was measured by VQA analysis of culture supernatants. (B) Method of analysis. IPDA analysis of each well provides the number of infected cells generated from a single infected cell plated at the beginning of the culture. RPP30 analysis of each well provides a measure of the total number of cells generated by the T cell proliferation that occurs in each well. The vast majority of these are uninfected cells (green), and thus the comparison of IPDA and RPP30 results provides an indication of the proliferative potential of infected cells relative to uninfected CD4⁺ T cells. Because the IPDA also provides an indication of proviral intactness, this comparison can be done for both cells with intact and defective proviruses. Similarly, measurement of supernatant virions in each well allows assessment of the effects of virus production on infected cell proliferation for cells with intact and defective proviruses. See Materials and methods for details and text for references.