Tim-3 expression is dysregulated in HIV-1 infection. (A) Flow cytometry gating strategy: Gates are set to include CD3- lymphocytes and exclude dead cells (viability marker+). NK cell subsets are defined by the expression of CD56 and CD16, and Tim-3+ NK cells by using appropriate fluorescence-minus-one controls. The four right flow cytometry panels show representative examples of Tim-3+ bulk and CD56bright, CD56dim and CD56neg NK cells in an HIV-1 negative subject (HIV-), an individual with late primary untreated infection, an untreated chronic HIV-1 progressor, and a HAART-treated HIV-1+ patient. Percentages of Tim-3+ bulk NK cells, and MFI of Tim-3 on CD56bright NK cells are indicated on the top left of the main panel and on the top right of the CD56bright NK cell gate, respectively. (B) Dot plots represent the percentage of Tim-3+ NK cells from 13 healthy individuals and 85 HIV-1-infected subjects (C), including 14 with early untreated HIV-1 infection, 54 with chronic untreated HIV-1 infection (blue, 17 viremic controllers; green, 17 elite controllers; red, 20 untreated progressors), and 17 with HAART-treated HIV-1 infection. (D) Percentages of Tim-3 + NK cells in CD56bright (CD3-CD56+CD16-), CD56dim (CD3-CD56+CD16+) and CD56neg (CD3-CD56-CD16+) NK cells in 13 HIV-1 negative and 85 HIV-1-infected subjects. (E) MFI of Tim-3 on CD56bright NK cells in 13 healthy individuals and 14 subjects with early infection, 54 with chronic untreated HIV-1 infection, including 17 viremic controllers (blue, VC), 17 elite controllers (green, EC), 20 untreated progressors (red, CU), and 17 with HAART-treated HIV-1 infection (CT). Horizontal lines indicate the median percentages. Statistical differences with p < 0.05 are indicated and were determined using the Mann–Whitney t test to compare phenotype frequencies between two groups.