Cytotoxic T lymphocytes and viral turnover in HIV type 1 infection

Abstract

To understand the role of the immune system in limiting HIV type 1 replication, it is critical to know to what extent the rapid turnover of productively infected cells is caused by viral cytopathicity or by immune-mediated lysis. We show that uncultured peripheral blood mononuclear cells of many patients contain cytotoxic T lymphocytes (CTL) that lyse target cells-at plausible peripheral blood mononuclear cell-to-target ratios-with half-lives of less than 1 day. In 23 patients with CD4 counts ranging from 10 to 900 per microliter, the average rate of CTL-mediated lysis corresponds to a target cell half-life of 0.7 day. We develop mathematical models to calculate the turnover rate of infected cells subjected to immune-mediated lysis and viral cytopathicity and to estimate the fraction of cells that are killed by CTL as opposed to virus. The models provide new interpretations of drug treatment dynamics and explain why the observed rate of virus decline is roughly constant for different patients. We conclude that in HIV type 1 infection, CTL-mediated lysis can reduce virus load by limiting virus production, with small effects on the half-life of infected cells.

Figure 1

The turnover rate of productively infected cells from 59 HIV-1-infected patients treated with antiviral drugs. The average death rate of productively infected cells, calculated from the exponential decline of plasma virus load after treatment, is 0.37 ± 0.18 per day, corresponding to a half-life of 1.9 days. The minimum half-life is about 1 day. There is no correlation between infected cell half-life and CD4 cell count. Symbols represent patients from different studies: ▵, ref. 1; ○, ref. 2; ×, ref. 4; □, ref. 5.

Figure 2

The average lifetime of target cells can be calculated from assays of specific cytotoxicity. (A) Time-resolved decay curve of peptide-sensitized target cells from HIV-1-infected, asymptomatic patient 84. The assays of fresh killing were performed using PBMC separated directly. Targets were HLA B8-matched B cells, untreated or prepulsed with 1 μM p17-3 (GGKKKYKL), an epitope in p17 to which this patient makes a predominant response. Targets were chromium-labeled, washed, and plated at 5000 cells per well in duplicate wells of a 96-well plate, and PBMC, medium, or Triton X (5%) was added to a total volume of 150 ml. Percentage-specific lysis was calculated as 100 × (experimental release − medium release)/(maximal release − medium release), and net specific lysis was determined by subtraction of background lysis against control targets. The decay rate of target cells can be directly obtained from the slope of the decay curves. There is an initial 2-hr time delay during which no killing can be observed. This may be a consequence of the effector cells needing time to attach to and kill the first targets. Between 2 and 8 hr, there is a roughly constant killing rate. PBMC-to-target ratios are 64:1 (○), 32:1 (▵), 16:1 (+), 8:1 (×), and 4:1 (◊). Different amounts of PBMC are added to the same number of targets. At a ratio of 64:1, target cell half-life is 11.6 hr (in the first 8 hr) and 8.7 hr (if calculated from the slope between 2 and 8 hr). (B) Half-life of target cells (calculated from 0 to 8 hr) versus PBMC-to-target ratio gives roughly a straight line in a double logarithmic plot with a slope of about −1. This suggests that the killing rate of target cells is simply proportional to the number of effector cells in the assay. (C) Half-life of HIV-infected target cells subjected to uncultured CTL-mediated lysis. Effector cells were freshly isolated PBMC from the B8-positive donor SC3. Targets were B8-matched C8166 cells used 48 hr after infection with 10 TCID₅₀ of HIV IIIB. Targets were chromium-labeled, washed, and plated at 5000 cells per well as described in A. (D) Half-lives of target cells derived from published studies of lysis by fresh PBMC from HIV-1-infected patients (–18). Points represent individual patients. (E) Half-life of target cells in assays of fresh cytotoxicity versus CD4 cell count of 23 patients. For each patient, we show the maximum response among the anti-Gag (○), anti-Pol (▵), or anti-Nef (×) CTL at a PBMC-to-target ratio of 50:1. (The anti-Tat response was never the maximum response in any of the patients.) The average rate of CTL-mediated lysis is 1.03 ± 0.57 per day, which corresponds to a half-life of 0.68 days. (At a PBMC-to-target ratio of 25:1, the average rate of CTL-mediated lysis is 0.62 ± 0.37 per day, which corresponds to a half-life of 1.1 day. Data not shown.) There is no correlation between CTL-mediated lysis and CD4 cell count. Effector cells were freshly isolated PBMC that were assessed for anti-HIV-1 CTL activity against ⁵¹Cr-labeled autologous B lymphoblastoid cell lines infected with recombinant vaccinia virus expressing HIV-1 (Lai) Gag, Pol, Env, Nef, or Tat at effector-to-target ratios of 50:1 and 25:1 (14, 15). Controls were target cells with medium alone for spontaneous release, and with detergent for maximal release of ⁵¹Cr. Spontaneous release averaged 17%. Split-sample validity testing showed <10% variation in CTL activity against the HIV-1 protein-expressing targets. Anti-HIV-1 CTL activity was not detected in PBMC from HIV-1 seronegative subjects.

Figure 3

A mathematical model to explore the effect of CTL-mediated killing on the half-life of infected cells and virus production: y₁ are newly infected cells that do not produce virus and are not killed by CTL, y₂ are cells that can be killed by CTL, and y₃ are cells that produce free virus and can be killed by CTL. The transition rates from y₁ to y₂ and from y₂ to y₃ are given by a(t) and b(t), respectively, where t is time since infection of the cell. The rate of CTL killing is given by α(t), and the rate of cell death due to virus is given by c(t). This is a general framework. We analyzed two specific cases. In model 1, we assumed that at time t₁ after infection, a cell becomes a target for CTL killing, and at time t₂, it starts to produce new virus particles. Death rate due to CTL killing is α, and death rate due to virus is c. These assumptions lead to an average lifetime of infected cells of T = t₁ + (1/α) − c/[α(α + c)] e^{−α(t₂−t₁)}. In the absence of CTL-mediated lysis (α = 0), the average lifetime is T = t₂ + (1/c). The of an infected cell is T_1/2 = t₁ + (1/α) log 2 if t₂ > t₁ + (1/α) log 2 or T_1/2 = t₂ + [1/(α + c)][log 2 − α(t₂ − t₁)] if t₂ < t₁ + (1/α) log 2. Note that the relation between half-life and average lifetime is not simply T_1/2 = T log 2, because infected cell death is a combination of different exponential declines. The average duration of a cell producing new virus is T_v = [1/(α + c)] e^{−α(t₂−t₁)}. In the absence of CTL, this increases to T₀ = 1/c. The fraction of cells that are killed by CTL before virus production sets in is f = 1 − e^{−α(t₂−t₁)}. The total fraction of cells killed by CTL as opposed to viral cytopathicity is F = 1 − [c/(α + c)] e^{−α(t₂−t₁)}. Assuming constant viral production after time t₂, the fraction of virus production inhibited by CTL-mediated lysis can be defined as 1 − T_v/T₀, which is equivalent to F. In model 2, we assumed that y₁ turns into y₂ at a constant rate, a; y₂ turns into y₃ at rate b; y₃ cells are killed by virus at rate c, and y₂ and y₃ cells are killed by CTL at rate α. In this model, the average lifetime of a cell is T = (1/a) + [1/(α + b)] + [b/(α + b)][1/(α + c)] and the average duration of a cell producing virus is T_v = [b/(α + b)][1/(α + c)]. In the absence of a CTL response, α = 0, this duration increases to T₀ = 1/c. The fraction of cells killed by CTL is F = 1 − [b/(α + b)][c/(α + c)], which is again equivalent to the total amount of virus production inhibited by CTL.

Figure 4

The models provide a new interpretation of the slope of virus decay in drug treatment studies (–6). We assume that before therapy, new infections occur at a constant rate β. This leads to an equilibrium distribution of y₁, y₂, and y₃ cells and free virus v. Drug treatment reduces β to zero, which leads to a decay of free virus and infected cells. (A) In model 1, the equilibrium distribution of infected cells is y₁(t) = 1 for t < t₁, y₂(t) = exp[−α(t − t₁)] for t₁ < t < t₂, and y₃(t) = exp[−α(t₂ − t₁) − (α + c)(t − t₂)] for t₂ < t, where t is the time since infection of the cell. Before drug treatment, the total amount of virus producing cells is Y₃(0) = ∫_t2^∞ y₃(t)dt = [1/(α + c)]exp[−a(t₂ − t₁)]. During drug treatment, this cell population declines as Y₃(T) = Y₃(0) for T < t₂, and Y₃(T) = Y₃(0)exp[−(α + c)(T − t₂)] for T > t₂. Here T denotes time after start of drug treatment. Thus, virus decline occurs with a shoulder of length t₂ followed by an exponential decline with slope α + c. (B) If we include a small fraction, h, of cells that are not exposed to CTL-mediated killing, the virus-producing cell population declines as Y₃(T) = [(1 − h)/(α + c)]exp[−α(t₂ − t₁) − (α + c)(T − t₂)] + (h/c)exp[−c(T − t₂)]. In a patient with a weak CTL response (α ≈ 0), the exponential decline is c, and in a patient with a strong CTL response (α ≫ c), the decline is again roughly c. Thus, the rate of virus decline does not reflect the rate of CTL-mediated killing, α. (C) In model 2, virus-producing cells, Y₃(T), decline as [(b − c)/a]e^−at + [(α − a + c)/(α + b)]e^−(α+b)t − [(α − a + b)/(α + c)]e^−(α+c)t. This expression again describes an initial shoulder followed by an exponential decay. The slope of the exponential decay is determined by the smallest value among a, b + α, or c + α. If the rate, a, at which infected cells proceed to become targets for CTL killing is slow, then the exponential decay in treatment studies may simply reflect this process and not depend on the rate of CTL-mediated killing, α. In all models, free virus is produced from infected cells according to v̇ = kY₃ − uv. If free virus turnover is fast, then v(T) is proportional to Y₃(T) and the decline of Y₃(T) can directly be interpreted as free virus decline; if not, then one more integration is necessary, but the conclusions are unaffected as long as u is not the slowest rate constant, which is very unlikely. For model 1, we chose c = 0.4, t₁ = 0.5, t₂ = 1, and h = 0.05. For model 2 we chose a = 0.4, b = 2, and c = 0.5 (continuous lines). Broken lines indicate noncytopathic virus with c = 0.01.