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HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells

Michael R. Betts, Martha C. Nason, Sadie M. West, Stephen C. De Rosa, Stephen A. Migueles, Jonathan Abraham, Michael M. Lederman, Jose M. Benito, Paul A. Goepfert, Mark Connors, Mario Roederer and Richard A. Koup

Article Figures & Data

Figures

  • Figure 1.

    Detection of 5 concurrent T-cell functions and characterization of HIV-specific CD8+ T-cell functionality in HIV-infected progressors. (A) Gating scheme for identification of multifunctional CD8+ T-cell responses. Shown are representative data of the HIV Gag-specific response from subject A26, an HIV-infected progressor, after a 6-hour in vitro stimulation. See “Patients, materials, and methods” for a detailed explanation of the procedure. (B) MIP-1β is the dominant HIV-specific CD8+ T-cell response (P < .001 for all stimulation conditions). Box plots represent the 10th, 25th, 50th, 75th, and 90th percentiles of the contribution of the indicated functional response (x-axis) toward the total CD8+ T-cell response against the indicated HIV peptide mix (color coded as shown) within the 79 progressors. The responses from each subject were standardized to allow comparison of the proportions of the total response expressing each function irrespective of the others. (C) Total CD8+ T-cell response frequency does not correlate with viral load. The red dots represent the total response summed across all functional combinations for each antigen for each progressor (n = 79). The x-axis denotes CD8+ T-cell frequency, and the y-axis denotes log10 viral RNA load. (D) The CD8+ T-cell response to HIV-Gag is composed of multiple functional subpopulations and is largely restricted to cell populations with limited functionality. The black bars represent the total CD8+ T-cell response frequency to Gag in subject A26 expressing the particular combination of functions shown. Each dot denotes CD107a, IFN-γ, MIP-1β, IL-2, and/or TNF-α positivity. The panel also contains horizontal bars of different colors showing those combinations of 5, 4, 3, 2, or 1 function for reference. Responses shown are background subtracted using the 28/49d negative control. (E) The functional profile of HIV-specific CD8+ T-cells is limited in HIV-infected progressors. Each pie chart represents the mean response across the 79 subjects to the 5 different HIV-antigen stimulations. For simplicity, responses are grouped by number of functions, matched to the colored bars in panel D. As shown, more than 75% of the average response to each antigen expresses fewer than 4 functions.

  • Figure 2.

    HIV-specific CD8+ T-cell functionality discriminates HIV-infected nonprogressors. (A) HIV Gag-specific CD8+ T-cell responses in HIV-infected nonprogressors are highly functional. The black bars represent the CD8+ T-cell response frequency to Gag in a representative nonprogressor (subject NP8) expressing the particular combination of functions shown. Each dot denotes CD107a, IFN-γ, MIP-1β, IL-2, and/or TNF-α positivity. The panel also contains horizontal bars of different colors showing those combinations of 5, 4, 3, 2, or 1 function for reference. Responses shown are background subtracted using the 28/49d negative control. (B) HIV-specific CD8+ T cells are highly functional in HIV-infected nonprogressors. Each pie chart represents the mean response across the 9 nonprogressors to the 5 different HIV-antigen stimulations. For simplicity, responses are grouped by number of functions, matched to the colored bars in panelA. As shown, responses with 5 functions can be detected to each HIV antigen in the nonprogressors, and a large proportion of the responding cells express 4 functions. (C) HIV-specific CD8+ T cells from nonprogressors (blue boxes) have a qualitatively different functional profile compared to progressors (red boxes). The box plots represent the 10th, 25th, 75th, and 90th percentiles of the proportion of the respective functional response toward the total CD8+ T-cell response against HIV Gag (left) or other HIV antigens (right panels). For simplicity, only the 5+ and 4+ populations lacking IL-2 production are shown for Pol, Env, Nef, and TRVVV responses; no differences were found for those functional combinations not shown. The responses from the cohorts were standardized so that the profiles could be compared irrespective of any frequency differences. Asterisks are placed above response pairs that are significantly different: ***P ≤ .001; **P ≤ .01. Marginal differences (P ≤ .05) are designated as a single asterisk. Each dot denotes a positive response for the function indicated at the bottom left.

  • Figure 3.

    The magnitude and proportion of the HIV-specific CD8+ T-cell response positive for all 5 functions is inversely correlated with viral load. Each dot represents data from a single progressor. The dotted line represents the linear regression (least-squares fit) line for predicting viral load based on the respective CD8+ T-cell response. Respective P values are shown in the top right of each graph. In the graphs, frequency refers to the magnitude of the antigen-specific response with a specific functional profile within the total CD8 pool, and percent refers to the proportion of the total antigen-specific response that bears a specific functional profile. The graphs depict: (A) frequency of 5+ Gag-specific cells versus viral load (slope =–30.6, r 2 = 0.15); (B) percent of Gag-specific response that is 5+ versus viral load (slope =–13.2, r 2 = 0.16); (C) percent of the total response (all antigens) that is 5+ versus viral load (slope =–23.8, r 2 = 0.19); (D) summed frequency of all 5+ responses from each antigen versus viral load (slope =–12.3, r 2 = 0.09); (E) the maximum frequency of any single 5+ response from any single antigen in each progressor versus viral load (each progressor is only represented by a single point; slope =–19.3, r 2 = 0.08); and (F) the percent of the Nef-specific response that is 5+ versus viral load (slope =–23.6, r 2 = 0.19). Note that the low r 2 values are primarily a function of the large number of values at 0 in the progressors, which cluster at higher viral loads.

  • Figure 4.

    Enhanced functionality in nonprogressors is not due to the presence of HLA-B57 or maintenance of central memory cells. (A) CMV-, EBV-, and influenzaspecific CD8+ T cells in the progressors are not restricted in functionality either off (left) or after initiation of ART (right). Each pie chart represents the average CD8+ T-cell response from a group of 5 progressors either prior to or after initiation of HAART. The colors correspond to the number of functions expressed, as shown. (B) HLA-B57+ (n = 6) and B57 (n = 3) nonprogressors were separated (left 2 pie charts), and the functionality of their HIV Gag-specific CD8+ T-cell responses compared with each other, and to HLA-B57+ (n = 6) and B57 (n = 6) progressors (right 2 pie charts). The colors in each pie chart correspond to the number of functions depicted by colored squares. (C) 5+ and (D) 4+ IL-2 responding cells can have varied memory phenotype. Twelve-color flow cytometry was performed to characterize the memory phenotype of responding CD8+ T cells. Plots depict CD8+ T cells responding with either the 5+ (red contours) or 4+IL-2 (orange contours) functional profile overlaid onto a density plot of the memory phenotype, as determined by CD27 and CD45RO, of the total CD8+ T-cell population. Inset in each plot is a histogram representing CD57 expression in the responding cells. (E) Initiation of ART does not improve CD8+ T-cell functionality in most individuals. Plots show the proportion of the total response against the indicated HIV peptide mix (indicated at top of each plot together with stimulus) prior to (OFF) and 6 to 12 months after (ON) initiation of ART. Each subject is represented by a colored line/symbol, and the symbols indicate the particular stimuli tested. The black box represents the mean response on or off therapy, with the SD of the mean shown as a black bar.

Tables

  • Table 1.

    Characteristics of HIV-infected patients

    PatientViral load, RNA copies/mLCD4 count, cells/mm3Therapy history*Sample month
    C1 27 000 466 Naive 42
    C2 19 000 273 Naive 33
    C3 < 50 677 3.9 y 222
    C4 18 000 398 Naive 6
    C5 708 000 514 3 y 118
    C6 59 000 361 Naive 13
    C7 17 000 636 1.3 y 91
    C8 71 000 391 Naive 7
    C9 400 718 Naive 83
    C10 44 000 428 Naive 25
    C11 18 000 508 Naive 21
    C12 22 000 384 Naive 117
    C13 14 000 413 Naive 167
    C14 21 000 693 Naive 29
    C15 35 000 478 Naive 14
    S1 2 350 368 4 mo 74
    S2 147 435 300 Unknown 43
    S3 26 918 810 6 mo 21
    S4 35 937 299 Naïve 0
    S5 150 ND 7 mo 76
    S6 500 000 209 Unknown 77
    S7 204 1056 Naive 27
    S8 190 700 Naive 16
    S9 1 054 684 Naive 23
    S10 10 366 162 Naive 0
    S11 54 194 459 Naive 0
    S12 172 852 196 Naive 1
    S13 2 153 450 Naive 0
    S14 73 780 690 Naive 12
    S15 70 414 406 Naive 1
    S16 15 315 700 Unknown 0
    S17 613 594 Naive 39
    S18 99 180 210 Naive 0
    S19 500 000 768 Naive 3
    S20 181 750 Naive 41
    S21 63 905 810 Naive 34
    S22 60 310 465 Naive 0
    S23 17 448 962 3 mo 51
    S24 706 1431 Naive 0
    S25 42 423 759 Unknown 0
    S26 8 941 671 Naive 0
    S27 62 948 783 Naive 6
    S28 69 668 850 Naive 0
    S29 10 694 1428 Naive 20
    A1 23 069 405 10 mo 182
    A2 4 917 1071 4 y 79
    A3 3 012 689 Naive 0
    A4 2 319 818 Naive 109
    A5 146 877 Naive 54
    A6 13 979 419 10 mo 21
    A7 72 662 570 Naive 209
    A8 185 720 22 Naive 1
    A9 41 459 167 3 y 63
    A10 148 000 36 Naive 219
    A11 18 993 286 Naive 1
    A12 49 755 372 Naive 21
    A13 5 107 716 3 y 131
    A14 377 759 1.3 y 176
    A15 24 985 252 Naive 99
    A16 10 391 563 1 y 90
    A17 48 680 294 1.9 y 63
    A18 16 121 531 2.5 y 59
    A19 47 551 838 1.3 y 76
    A20 65 548 388 Naive 101
    A21 103 503 Naive 30
    A22 63 382 426 Naive 13
    A23 128 540 431 1.5 y 123
    A24 68 380 150 8 mo 95
    A25 6 369 857 Naive 119
    A26 659 619 Naive 139
    A27 19 700 542 3 y 80
    A28 2 981 1049 Naive 189
    A29 30 553 835 Naive 27
    A30 64 975 Naive 172
    A31 21 159 571 1.8 y 108
    A32 5 821 694 Naive 10
    A33 239 300 4 Naive 58
    A34 128 460 228 Naive 136
    A35 10 333 513 Naive 41
    NP1 124 1830 Naive 122
    NP2 < 50 311 Naive 219
    NP3 < 50 908 Naive 216
    NP4 < 50 1042 Naive 222
    NP5 < 50 1645 Naive 165
    NP6 59 972 Naive 103
    NP7 < 50 785 Naive 72
    NP8 < 50 1506 Naive 179
    NP9 < 50 806 Naive 57
    • ND indicates not determined.

    • * Time since most recent documented ART. All patients were off therapy at the time of sampling. Patients with undocumented therapy history prior to sampling are listed as unknown.

    • Sample month refers to the time between HIV diagnosis and the sampling date. For patients S16 and S25, the diagnosis date was unknown, so a value of 0 was used.