Volume 10, Issue 6, Pages 661-671 (June 1999) The Selective Downregulation of Class I Major Histocompatibility Complex Proteins by HIV-1 Protects HIV-Infected Cells from NK Cells  George B Cohen, Rajesh T Gandhi, Daniel M Davis, Ofer Mandelboim, Benjamin K Chen, Jack L Strominger, David Baltimore  Immunity  Volume 10, Issue 6, Pages 661-671 (June 1999) DOI: 10.1016/S1074-7613(00)80065-5

Figure 1 HIV-1 Selectively Downregulates Class I MHC Proteins 221 cells stably expressing defined class I proteins were infected with the NL-PI reporter virus. At 40–48 hr post infection, these cells were stained with a PLAP mAb (x axis) and a pan-class I MHC mAb (y axis). The NL-PI reporter virus used to infect these cells carries the full complement of HIV-1 genes except for (B) and (C), which used, respectively, a Nef-minus and Vpu-minus NL-PI virus. The infected 221 cells expressed (A) HLA-A2, (B) HLA-A2, (C) HLA-A2, (D) HLA-B2705, and (E) HLA-Cw4. In (F), we infect C1R cells. Immunity 1999 10, 661-671DOI: (10.1016/S1074-7613(00)80065-5)

Figure 2 Region on Class I MHC Molecules Required for Downregulation by HIV-1 221 cells expressing class I chimeric molecules were infected with the NL-PI virus. The cells were stained 40–48 hr post infection with a PLAP mAb (x axis) and either a CD8 mAb (2A-2B; y axis) or a pan-class I MHC mAb (2C-2F; y axis). The infected 221 cells expressed (A) CD8α, (B) CD8α/A2(Tm&tail), (C) HLA-Cw4/A2(Tm&tail), (D) HLA-Cw4/A2-tail, (E) HLA-Cw4/E(Tm&tail), and (F) HLA-A2/E(Tm&tail). Immunity 1999 10, 661-671DOI: (10.1016/S1074-7613(00)80065-5)

Figure 3 Identification of Residues in the Cytoplasmic Tail Region of Class I MHC Proteins that Are Critical for Nef-Dependent Downregulation 221 cells expressing the indicated class I allotypes were infected with the NL-PI virus and stained for PLAP (x axis) and class I MHC (y axis) 40–48 hr post infection. The infected 221 cells expressed (A) HLA-Cw4, (B) -Cw4/B27-tail, (C) -Cw4/B27(Y321C), and (D) -Cw4(C321Y). In (E)–(J), HLA-Cw4(C321Y ΔC3) was further mutated to make -Cw4 progressively more like HLA-B with the following mutations: (E) I338T; (F) E335V; (G) N328D; (H) E335V and I338T; (I) N328D and I338T; and (J) N328D and E335V. In (K) and (L), the HLA-Cw4/E-tail chimera was made more like the class I consensus cytoplasmic tail with the following mutations: (K) HLA-Cw4/E (QAS) contains K323Q, E325A, and W326S; and (L) HLA-Cw4/E (E325A). Immunity 1999 10, 661-671DOI: (10.1016/S1074-7613(00)80065-5)

Figure 4 HLA-Cw4 Protects HIV-Infected Cells from NK Cell Lysis 221 cells expressing HLA-A2 alone (column 1), -A2 and -Cw3 (column 2), and -A2 and -Cw4 (column 3) were infected with HIV carrying a GFP reporter, NL-GI. At 35–45 hr post infection, the infected cells were mixed with either YTS.NKIR1 cells (A and B), NK.KIR1 cells (C and D), or YTS cells (E) at a 4:1 effector:target cell ratio. Cells were either stained immediately (time = 0 hr) for GFP (x axis) and HLA-A2 (y axis) (A) and (C) or incubated for 6 hr at 37°C (time = 6 hr) and then stained (B, D, and E). Not shown are YTS cells mixed with target cells at time 0 hr as these looked identical to the YTS.NKIR1 cells at 0 hr (A). YTS and YTS.NKIR1 cells are uninfected and HLA-A2-negative and therefore appear near the origin (lower left quadrant) in (A), (B), and (E). The cells shown in Figures 4; 5; 6 have been electronically gated based on light scattering to exclude dead cells. Primary NK.NKIR1 cells are much smaller than 221 cells and have mostly been excluded from the analysis presented in (C) and (D) based on light-scattering properties. To facilitate comparisons of the ability of NK cells to selectively kill HIV-infected cells, the proportion of 221.A2.Cw4 cells (column 3) that are uninfected (upper left quadrant), infected but have not yet downregulated HLA-A2 (upper right quadrant), or infected and have downregulated HLA-A2 (lower right quadrant) are reported in (A)–(D). No such numbers are reported for 221.A2.Cw4 cells mixed with YTS cells (E), as too few cells survive to make the numbers meaningful. Immunity 1999 10, 661-671DOI: (10.1016/S1074-7613(00)80065-5)

Figure 5 NK.NKIR1 Cells Can Selectively Kill 221 Cells that Downregulate a HLA-Cw4/A2-Tail Chimera 221 cells expressing either -Cw4 (column 1) or the -Cw4/A2-tail chimera (column 2) were infected with the NL-GI virus and mixed with the primary NK.NKIR1 cells. Cells were either stained immediately (time = 0 hr) for GFP (x axis) and class I MHC protein (y axis) (A) or incubated for 6 hr at 37°C (time = 6 hr) and then stained (B). Primary NK.NKIR1 cells are much smaller than 221 cells and have been excluded from the analysis presented based on light-scattering properties. To facilitate comparisons of the ability of NK cells to kill HIV-infected cells, the proportion of 221.Cw4 and 221.A2.Cw4 cells are reported in the respective quadrants. Immunity 1999 10, 661-671DOI: (10.1016/S1074-7613(00)80065-5)

Figure 6 HLA-E Protects HIV-Infected Cells from NK Cell Lysis 221 cells expressing HLA-B2705 (column 1), HLA-Cw3 (column 2), and HLA-A2 leader/E (column 3) were infected with HIV carrying a GFP reporter, NL-GI. At 35–45 hr post infection, the infected cells were analyzed for class I downregulation (A) or mixed with NK.CD94 cells (B–D) at effector to target cells ratios of 5:1–10:1. Cells were either stained immediately (time = 0 hr) for GFP (x axis) and MHC class I (y axis) (B) or incubated for 6 hr at 37°C (time = 6 hr) and then stained (C and D). NK cells in (D) were preincubated with an anti-CD94 mAb to block the CD94/NKG2A interaction with HLA-E. Primary NK.CD94 cells are much smaller than 221 cells and have mostly been excluded from the analysis presented in (B)–(D) based on light-scattering properties. To facilitate comparisons of the ability of NK cells to selectively kill HIV-infected cells, the proportion of HIV-infected cells are reported in (C) and (D). No such numbers are reported when too few cells survive to make the numbers meaningful. Immunity 1999 10, 661-671DOI: (10.1016/S1074-7613(00)80065-5)