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Keywords:

  • CD8/cytotoxic T cells;
  • human immunodeficiency virus (HIV);
  • interleukin-7 (IL-7);
  • memory response

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Memory CD8+ T cells regain function during a recall response, but the requirement of signals in addition to antigen during a secondary immune response is unknown. In this study, the ability of interleukin-7 (IL-7) to enhance memory CD8CD45RA− CD127+ T-cell responses in health and in human immunodeficiency virus (HIV) infection was investigated. CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMCs) from HIV and untreated HIV+ donors were pulsed with a cytomegalovirus/Epstein–Barr virus/influenza (CEF) peptide pool, and co-cultured with autologous memory CD8+ T cells in the presence of IL-7. Cell survival and the function of memory CD8+ T-cell subsets were then evaluated. Memory CD8+ T-cell proliferation and interferon-γ (IFN-γ) production was enhanced by the presence of antigen, and the addition of IL-7 further enhanced antigen-induced proliferation. In HIV+ individuals, the presence of antigen enhanced IFN-γ production to a small degree but did not enhance proliferation. Lastly, IL-7 did not enhance antigen-mediated proliferation of memory CD8+ T cells from HIV+ individuals. IL-7 therefore appears to have a role in secondary immune responses and its activity is impaired in memory CD8+ T cells from HIV+ individuals. These results further our understanding of the signals involved in secondary immune responses, and provide new insight into the loss of CD8+ T-cell function in HIV infection.


Abbreviations:
ART

antiretroviral therapy

BSA

bovine serum albumin

CEF

cytomegalovirus/Epstein–Barr virus/influenza

CFSE

carboxyfluorescein diacetate succinimidyl ester

CMV

cytomegalovirus

DMSO

dimethylsulphoxide

EBV

Epstein–Barr virus

FCS

fetal calf serum

FITC

fluorescein isothiocyanate

HAART

highly active antiretroviral therapy

HIV

human immunodeficiency virus

IFN-α

interferon-α

IL

interleukin

PBMCs

peripheral blood mononuclear cells

PBS

phosphate-buffered saline

PE

phycoerythrin

PMA

phorbol 12-myristate 13-acetate

TCM

central memory CD8+ T cell

TEM

effector memory CD8+ T cell

TEMRA

terminally differentiated effector memory CD8+ T cell

TN

naïve CD8+ T cell

TNF-α

tumour necrosis factor-α

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

During a primary immune response, a proportion of antigen-specific naïve CD8+ T cells differentiate into cytotoxic effector cells. Following the effector phase, most effector cells die during the contraction phase, yet long-lasting memory CD8+ T cells remain. Current models of differentiation describe either a linear development of memory cells directly from effector CD8+ T cells, or a non-linear development of memory cells directly from naïve and effector CD8+ T cells.1–4 Upon re-encounter with antigen, central memory (TCM, CD45RA− CCR7+) and effector memory (TEM, CD45RA− CCR7) CD8+ T cells exhibit effector functions including proliferation, perforin production, degranulation and production of cytokines [interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin (IL)-2]5,6 and are termed polyfunctional when a single memory CD8+ T cell is capable of multiple functions. Secondary immune responses are typically generated faster, and are of a greater magnitude, than primary immune responses. This is because memory CD8+ T cells proliferate at a higher rate than naïve CD8+ T cells and require a lower threshold of antigenic stimulation.7–9 Effector memory CD8+ T cells can differentiate into terminally differentiated effector memory (TEMRA, CD45RACCR7) CD8+ T cells, and their cytolytic functions increase as they differentiate.10 The mechanisms required for an effective secondary immune response are still unknown. Many cytokines, particularly those of the IL-2 receptor gamma (IL-2Rγ)-chain sharing cytokine family (IL-2, -4, -7, -15 and -21), can affect CD8+ T-cell responses, but the role of these cytokines in enhancing the function of memory CD8+ T cells during a secondary immune response has not been fully characterized.

IL-7 signals through the IL-7 receptor complex, which is composed of the IL-7Rα chain (CD127) and the common IL-2Rγ chain (CD132), and is critical for early T-cell development and homeostasis of naïve and memory CD8+ T cells.11 IL-7 has been shown to increase the proliferation of CD4+ and CD8+ T cells in vitro and in vivo.12–14 As cells differentiate from naïve to effector CD8+ T cells, CD127 expression is down-regulated.15 In a non-linear model of differentiation, IL-7 enhances the survival of memory cells that did not pass through an effector phase.16,17 IL-7 has also been shown to enhance the effector functions of human immunodeficiency virus (HIV)-specific CD8+ T cells18, as well as to reduce the spontaneous apoptosis of CD4+ and CD8+ T cells during HIV infection.19

In progressive HIV infection, CD8+ T-cell function becomes increasingly impaired. The reasons for this loss of function are not known; however, several factors may be involved, including chronic immune activation, decreased CD4+ T-cell help, or decreased cytokine responsiveness.20–22 Cytokine production is dysregulated in HIV infection, and, in particular, the plasma IL-7 concentrations are increased.23–25 Despite increased plasma concentrations of IL-7, CD4+ and CD8+ T cells appear to be less responsive to IL-7 stimulation,22,26–28 which may contribute to the observed CD8+ T-cell dysfunction. This could be caused by the observed downregulation of CD127 expression on CD8+ T cells,25,29–32 or an inherent cellular impairment of IL-7 activity. IL-7 activity is important for survival and differentiation during a primary immune response, is critical for maintenance of the peripheral CD8+ T-cell population and is required for memory CD8+ T-cell generation and survival. It is therefore possible that IL-7 also enhances CD8+ T-cell function during a secondary immune response. We hypothesized that IL-7 would enhance CD8+ T-cell memory responses and that this activity is impaired in HIV infection. To do so, we evaluated the effects of antigen stimulation, with or without IL-7, on CD8CD45RA− CD127+ memory T cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Memory CD8+ T-cell isolation

Blood was drawn from HIV and HIV+ individuals after obtaining written informed consent. All aspects of this study were approved by the Ottawa Hospital Research Ethics Board. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll–Hypaque density-gradient centrifugation. Untouched CD8+ T cells were isolated by negative selection from PBMCs by magnetic cell sorting using the CD8+ T cell Isolation Kit (Miltenyi Biotec, Auburn, CA) and the ‘deplete’ program on an autoMACS separator (Miltenyi Biotec). This process yielded a CD8+ T-cell-depleted population of PBMCs, as well as a CD8+ T-cell population that could be further sorted into memory subsets. After separation, CD8+ T-cell purity was approximately 98%. Isolated cells were cultured in complete RPMI (Invitrogen, Burlington, ON, Canada) supplemented with 20% fetal calf serum (FCS; Invitrogen) and 1% penicillin/streptomycin (Invitrogen) at 37° and 5% CO2. After overnight incubation, CD8+ T cells were washed and CD8CD45RA T cells were isolated by magnetic cell sorting using CD45RA microbeads (Miltenyi Biotec) and the ‘possel’ program on an autoMACS separator (Miltenyi Biotech), achieving a purity of 99·10 ± 0·59%. From this fraction, the CD127+ memory CD8+ T cells were obtained by magnetic cell separation using phycoerythrin (PE)-conjugated CD127 antibodies (Beckman Coulter, Mississauga, ON, Canada) and anti-PE microbeads (Miltenyi Biotec), and the ‘possels’ program on an autoMACS separator (Miltenyi Biotec). The resulting CD8CD45RA− CD127+ T cells, of 96·17 ± 5·61% purity, were maintained in complete RPMI (Invitrogen) until the initiation of co-culture. Isolation of CD45RA T cells allowed the direct study of memory cell function, and the isolation of CD127+ T cells controlled for the fact that in HIV+ individuals this population of cells is under-represented.

Cell co-culture and antigen stimulation

CD8+ T-cell-depleted PBMCs were washed and pulsed with a cytomegalovirus/Epstein–Barr virus/influenza (CEF) peptide pool [i.e. containing common antigens to influenza, Epstein–Barr virus (EBV) and cytomegalovirus (CMV); PANATecs GmbH, Tubingen, Germany] and incubated at 37° for 90 min. After the incubation, the CD8+ T-cell-depleted PBMCs were washed with phosphate-buffered saline (PBS) and resuspended in complete RPMI (Invitrogen). CD8CD45RA− CD127+ T cells were co-cultured with peptide-pulsed autologous CD8+ T-cell-depleted PBMCs at a 1:5 ratio in 96-well plates. CD8CD45RA− CD127+ T cells were cultured with unpulsed CD8+ T-cell-depleted PBMCs to determine background proliferation and function in the absence of stimulation. Cells were also cultured with increasing concentrations of IL-7 (1, 10, or 50 ng/ml; R&D Systems, Minneapolis, MN) for either 6 days to analyze proliferation or 6 hr to evaluate IFN-γ production and CD107a/b expression.

Proliferation of memory CD8+ T cells

To investigate the proliferation of memory CD8+ T cells, carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) dilution was analyzed by flow cytometry. A 5 mm CFSE stock solution in dimethylsulphoxide (DMSO; Sigma Aldrich, Oakville, ON, Canada) was used, and a 12 μm working solution of CFSE was prepared containing 0·1% bovine serum albumin (BSA; Sigma Aldrich). CD8CD45RA− CD127+ T cells were washed in PBS and resuspended in the CFSE working solution at a concentration of 107 cells/ml and then incubated at 37° for 10 min. After incubation, staining was quenched with cold RPMI (Invitrogen) and the cells were incubated on ice for 5 min. The cells were then centrifuged and resuspended at 1 × 106 cells/ml in complete RPMI. The cells were co-cultured for 6 days, and CFSE dilution was analyzed using flow cytometry. Compensation was performed manually using both single-positive and double-positive control samples, following a fluorescence minus one compensation strategy. Data generated from flow cytometry analyses were analyzed after acquisition using FCS Express, version 2.0 (De Novo Software, Los Angeles, CA). Analysis of CFSE+ cells included cells contained in a live cell gate based on forward-scatter and side-scatter distributions, as described previously,33 as well as on CFSE+ cells. CD8+ T-cell subsets were distinguished using phycoerythrin-Texas red energy coupled dye (ECD)-conjugated CD45RA antibodies (Beckman Coulter) and phycoerythrin-Cy7 (PC7)-conjugated CCR7 antibodies (BD Biosciences, Mississauga, ON, Canada). CFSElo cells were defined as cells that had undergone three or more divisions, as described previously.33

Analysis of the function of CD8+ T cells

To assess the function of memory CD8+ T cells, the production of IFN-γ and the expression of CD107a/b, a marker of degranulation, were measured. Memory CD8+ T cells were co-cultured, as described above, in 96-well round-bottom plates with peptide-pulsed or unpulsed CD8+ T-cell-depleted PBMCs and increasing concentrations of IL-7 (1–50 ng/ml), along with anti-CD28/anti-CD49d Ig (Ms IgG2b/Ms IgG1) (BD Biosciences) as required for costimulation. To measure CD107a/b expression, fluorescein isothiocyanate (FITC)-conjugated CD107a/b antibodies (BD Biosciences) were added at the onset of culture. Cells were co-cultured for 1 hr at 37°, after which brefeldin A (10 μg/ml) was added for 5 hr at 37° to stop protein secretion. Cells were fixed and permeabilized using the Fix and Perm kit (Invitrogen) and analyzed for IFN-γ production using FITC-conjugated IFN-γ antibodies (Beckman Coulter) or for CD107a/b expression by flow cytometry. CD8+ T-cell subsets were distinguished, as described above, using the antibody conjugates CD45RA–ECD and CCR7–PC7, as well as PC5-conjugated CD8 antibodies (Beckman Coulter). As a positive control, cells were cultured with phorbol 12-myristate 13-acetate (PMA) (0·005 μg/ml; Sigma Aldrich) and ionomycin (0·5 μg/ml; Sigma Aldrich) to ensure that the assay was effective in detecting increased IFN-γ production and CD107a/b expression (data not shown).

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 4.0 software (Graph Pad Software, San Diego, CA), and statistical significance was determined by using either the one-tailed paired Student’s t-test or a one-way analysis of variance (anova) followed by Dunnett’s post-test, as appropriate.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Cell phenotype and gating

To directly evaluate the responsiveness of memory CD8+ T cells, CD8CD45RA− CD127+ T cells were isolated from PBMCs. This enabled the study of a specific cell population with the potential to respond to IL-7. Furthermore, isolation of a CD127+ cell subset allowed the direct study and interpretation of IL-7 activity, even in individuals with a low proportion of CD8CD127+ T cells, as is the case in HIV+ individuals. In HIV individuals, CD127 was expressed on approximately 80% of CD8+ T cells (data not shown), which is consistent with previous findings.25,29,32,34,35 The proportion of T-cell subsets in bulk CD8+ T cells of healthy individuals was as follows: naïve CD8+ T cell (TN) (52·5%) > TCM (17·9%) > TEM (15·5%) > TEMRA (14·3%) (Fig. 1a). After isolating CD8CD45RA− CD127+ T cells, the initial distribution of memory CD8+ T-cell subsets was TCM (68·9%) > TEM (30·4%) (Fig. 1b).

image

Figure 1.  CD8+ T-cell subset distribution. Representative dot-plots are shown of bulk CD8+ T cells isolated from the peripheral blood mononuclear cells (PBMCs) of (a) human immunodeficiency virus-negative (HIV) individuals and (c) human immunodeficiency virus-positive (HIV+) individuals, and CD45RA and CCR7 expression was analyzed to determine cell subset distribution. CD8CD45RA− CD127+ T cells were isolated from bulk CD8+ T cells from (b) HIV individuals and (d) HIV+ individuals and stained for CD45RA and CCR7 to determine initial memory CD8+ T-cell subset distribution.

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IL-7 enhances antigen-mediated proliferation in memory CD8+ T cells

The effect of IL-7 on antigen-mediated proliferation of isolated CD8CD45RA− CD127+ T cells was evaluated. CD8CD45RA− CD127+ T cells were stained with CFSE and co-cultured with autologous peptide-pulsed PBMCs and increasing concentrations of IL-7 (1–50 ng/ml) for 6 days. The proportion of dividing cells (the percentage of CFSElo) and the expression of CD45RA and CCR7 were then analyzed using flow cytometry. Exposure to antigen resulted in an increased proliferation of CD8CD45RA− CD127+ T cells (P = 0·012, determined using the Student’s t-test; Fig. 2a), specifically in the TCM and TEM cell subsets (= 0·022 and = 0·033, respectively, determined using the Student’s t-test; Fig. 2b). The TEMRA subset, which represents memory CD8+ T cells that have differentiated from the initial population, proliferated to the greatest extent, regardless of antigen stimulation (Fig. 2b). Cells cultured with IL-7 alone, at concentrations of 10 and 50 ng/ml, enhanced the proliferation of CD8CD45RA− CD127+ T cells (data not shown), and the addition of IL-7 at 10 and 50 ng/ml further enhanced the antigen-mediated proliferation of CD8CD45RA− CD127+ T cells (= 0·002, determined using anova; and P < 0·01, determined using Dunnett’s post-test; Fig. 2c), specifically in the TCM (= 0·002, determined using anova; and P < 0·01, determined using Dunnett’s post-test) and TEM (P < 0·0001, determined using anova; and P < 0·01, determined using Dunnett’s post-test) subsets (Fig. 2d). Analyzing the data with the proportion of proliferating cells being defined as those undergoing one or more divisions yielded similar results.

image

Figure 2.  Antigen and interleukin-7 (IL-7) increased the proliferation of CD8CD45RA− CD127+ T cells. CD8CD45RA− CD127+ T cells were co-cultured with peptide-pulsed CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMCs) for 6 days, and then carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution, CD45RA expression and CCR7 expression were analyzed by flow cytometry and gated on CFSE+ cells. Proliferation was enhanced by the presence of antigen in (a) total memory CD8+ T cells (*P = 0·012, determined using the Student’s t-test), as shown in a representative dot-plot summarized in a bar graph. (b) These effects occurred specifically in the central memory CD8+ T cell (TCM) and effector memory CD8+ T cell (TEM) subsets (**P = 0·022 and ***P = 0·033, respectively, determined using the Student’s t-test). IL-7 enhanced antigen-mediated proliferation of (c) total memory CD8+ T cells [*P = 0·004, determined using analysis of variance (anova); and P < 0·05, determined using Dunnett’s post-test] and (d) memory CD8+ T-cell subsets, specifically the TCM (**P = 0·0001, determined using anova; and P < 0·01, determined using Dunnett’s post-test) and TEM (***P = 0·0005, determined using anova; and P < 0·01, determined using Dunnett’s post-test) subsets (= 6). TEMRA, terminally differentiated effector memory CD8+ T-cell subset.

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IL-7 does not enhance IFN-γ expression or CD107a/b expression in memory CD8CD127+ T cells

To evaluate the effect of IL-7 on the antigen-mediated function of CD8CD45RA− CD127+ T cells, IFN-γ production was analyzed. CD8CD45RA− CD127+ T cells were co-cultured for 6 hr with peptide-pulsed CD8+ T-cell-depleted PBMCs, with or without IL-7. Cells were then fixed and permeabilized, and analyzed for IFN-γ, CD45RA and CCR7 expression. In the presence of CEF antigen, IFN-γ production was increased (= 0·023, determined using the Student’s t-test; Fig. 3a), specifically in the TEM (= 0·023, determined using the Student’s t-test) and TEMRA (= 0·031, determined using the Student’s t-test) subsets (Fig. 3b). Unlike that observed with proliferation, the addition of IL-7 did not enhance antigen-induced IFN-γ expression by CD8+ CD45RACD127+ T cells (Fig. 3c,d).

image

Figure 3.  Antigen-induced interferon-γ (IFN-γ) production by CD8+ CD45RACD127+ T cells. CD8+ CD45RACD127+ T cells were co-cultured with peptide-pulsed CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMCs) for 6 hr and then IFN-γ production, and CD8, CD45RA and CCR7 expression, were analyzed by flow cytometry. Representative dot-plots show antigen-enhanced IFN-γ production by (a) total memory CD8+ T cells (*P = 0·023, determined using the Student’s t-test) and the data are summarized in bar graphs. (b) Antigen specifically induced IFN-γ expression in individual memory CD8+ T-cell subsets, specifically the effector memory CD8+ T cell (TEM) and terminally differentiated effector memory CD8+ T cell (TEMRA) subsets (**P = 0·0008 and ***P = 0·031, determined using the Student’s t-test). Interleukin-7 (IL-7) did not enhance antigen-mediated IFN-γ production of (c) total memory CD8+ T cells or (d) memory CD8+ T-cell subsets (= 6). TCM, central memory CD8+ T-cell subset.

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To evaluate another aspect of CD8+ T-cell function, the surface expression of CD107a/b was analyzed. The CD107a/b molecules are localized in intracellular vesicles and are subsequently expressed on the surface when cells degranulate.36 CD8CD45RA− CD127+ T cells were co-cultured with peptide-pulsed CD8+ T-cell-depleted PBMCs, with or without IL-7 for 6 hr, after which the cells were fixed, permeabilized and analyzed for CD107a/b, CD45RA and CCR7 expression. The presence of antigen did not increase the expression of CD107a/b on CD8CD45RA− CD127+ T cells in any memory CD8+ T-cell subset (Fig. 4a,b). The addition of IL-7 had no impact on CD107a/b expression in antigen-stimulated CD8CD45RA− CD127+ T cells (Fig. 4c,d).

image

Figure 4.  Neither antigen nor interleukin-7 (IL-7) induced CD107a/b expression on CD8CD45RA− CD127+ T cells. CD8CD45RA− CD127+ T cells were co-cultured with peptide-pulsed CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMCs) for 6 hr, and then the expression of CD107a/b CD8, CD45RA and CCR7 were analyzed by flow cytometry. Representative dot-plots show how antigen did not induce CD107a/b expression on (a) total memory CD8+ T cells or (b) the memory CD8+ T-cell subsets, and data are summarized in bar graphs. IL-7 did not enhance antigen-mediated CD107a/b expression on (c) total memory CD8+ T cells or (d) memory CD8+ T-cell subsets (n = 6). TCM, central memory CD8+ T-cell subset; TEM, effector memory CD8+ T-cell subset; TEMRA, terminally differentiated effector memory CD8+ T-cell subset.

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Memory CD8+ T-cell distribution is skewed in HIV+ individuals

To determine the effect of antigen and IL-7 on the proliferation and function of memory CD8+ T cells during HIV infection, CD8+ T cells were isolated from HIV+ individuals. HIV+ individuals were either naïve to therapy or had been off antiretroviral therapy (ART) for longer than 1 year, with mean CD4+ T-cell counts of 259·2 cells/ml and mean viral loads of 4·53 log10 copies/ml (Table 1). CD127 was expressed on approximately 20% of CD8+ T cells from HIV+ individuals (data not shown), significantly lower than that observed in HIV individuals and consistent with previous findings.29,32 The proportion of T-cell subsets in bulk CD8+ T cells differed between HIV (Fig. 1a) and HIV+ (Fig. 1c) individuals before the isolation of memory cells. There were significantly fewer TN cells and TCM cells, and more TEMRA and TEM cells, in HIV+ individuals than in HIV individuals (Table 2), consistent with previous reports.37,38 After isolating CD8CD45RA− CD127+ T cells, the subset distribution was TCM (65·0%) > TEM (34·6%) (Fig. 1d), which is comparable to that observed in HIV individuals (Fig. 1b).

Table 1.   Characteristics of human immunodeficiency virus-positive (HIV+) patients
 CharacteristicMean (range)
  1. = 10.

  2. 1ART, antiretroviral therapy.

Age (years)42·5 (33.0–60.0)
CD4 count (cells/μl)259·2 (65.0–499.0)
CD4 count (%)15·6 (8·5–28·5)
Viral load (log copies/ml)4·53 (3·91–5·28)
Treatment history8 naïve; 2 off ARV1 for > 1 year
Table 2.   CD8+ T-cell subsets from human immunodeficiency virus-negative (HIV) and human immunodeficiency virus-positive (HIV+) individuals
CD8+ T-cell subsetsHIV individualsHIV+ individuals
  1. n = 10 in each group.

  2. The results are expressed as mean (range).

  3. TCM, central memory; TEM, effector memory; TEMRA, terminally differentiated effector memory; TN, naïve.

% TN (CD45RACCR7+)45·4 (36–53·9)19·8 (19·3–20·5)
% TCM (CD45RA− CCR7+)21·5 (14–29·3)10·6 (4·7–14·7)
% TEM (CD45RA− CCR7)19 .0 (14·5–23·8)23·5 (12·0–33·0)
% TEMRA (CD45RACCR7)14·2 (10·6–20·2)46·2 (31·8–64·1)

Memory CD8+ T-cell function and IL-7 responsiveness is impaired during HIV infection

To evaluate the effect of IL-7 on memory CD8CD127+ T-cell function during HIV infection, the proliferation, IFN-γ production and CD107a/b expression in response to antigen and IL-7 was analyzed. CD8+ CD45RACD127+ T cells from HIV+ individuals did not proliferate in response to CEF antigens in any memory CD8+ T-cell subset (Fig. 5a). In response to antigen, IFN-γ production by CD8CD45RA− CD127+ T cells was increased (P = 0·030, determined using the Student’s t-test; Fig. 5b), specifically in the TEM and TEMRA subsets (P = 0·050 and P = 0·024, respectively, determined using the Student’s t-test; data not shown); however, IFN-γ production was lower than that observed in HIV individuals (P = 0·058 for total memory CD8+ T cells, determined using the Student’s t-test). Degranulation, as measured by CD107a/b expression, was not enhanced following exposure to antigen (Fig. 5c). When memory CD8CD45RA− C127+ T cells from HIV+ individuals were cultured with IL-7, proliferation was not enhanced (Fig. 5a), in contrast to that observed in CD8+ T cells from HIV individuals (Fig. 2). Neither antigen-mediated IFN-γ production nor CD107a/b expression was enhanced by IL-7 in HIV+ individuals (data not shown) similar to the findings in HIV individuals (Fig. 3).

image

Figure 5.  Neither antigen nor interleukin-7 (IL-7) enhanced the function of CD8CD45RA− CD127+ T cells from human immunodeficiency virus-positive (HIV+) individuals. CD8CD45RA− CD127+ T cells from HIV+ individuals were co-cultured with peptide-pulsed CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMCs) and analyzed for functional responses by flow cytometry. (a) Cells were cultured for 6 days with antigen alone or with antigen and IL-7 (10 ng/ml), and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution was analyzed by flow cytometry and gated on CFSE+ cells. CD8CD45RA− CD127+ T cells were also co-cultured for 6 hr with antigen and it was found that (b) interferon-γ (IFN-γ) production was enhanced in total memory CD8+ T cells (*P = 0·030, determined using the Student’s t-test), and (c) CD107a/b expression was not enhanced (= 5).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Upon re-exposure to antigen, memory CD8+ T cells exhibit a number of functions that characterize a secondary immune response. Although IL-7 is known to have a role in memory T-cell development and survival,15,39–41 its role in enhancing recall responses is otherwise unknown. This was directly investigated in the present study by isolating CD8CD45RA− CD127+ T cells. As anticipated, antigen stimulation enhanced the proliferation of memory CD8+ T cells in the TCM and the TEM subsets (Fig. 2b). To evaluate the potential ability of IL-7 to enhance antigen-mediated proliferation, CD8CD45RA− CD127+ T cells were co-cultured with antigen-pulsed CD8+ T-cell-depleted PBMCs and increasing concentrations of IL-7. Proliferation of TCM and TEM CD8+ T cells was enhanced by IL-7 (Fig. 2d), suggesting that this cytokine plays an important role in a memory immune response. In contrast to its effect on antigen-mediated proliferation, IL-7 did not enhance antigen-mediated IFN-γ production by CD8CD45RA− CD127+ T cells (Fig. 3c). Previous studies have shown that IL-7 can enhance IFN-γ production in activated T cells in vitro;42 however, this was observed in a mixed population of CD4+ and CD8+ T cells, and IL-7 may affect the production of IFN-γ by naïve and memory T cells differently. IL-7 may also act synergistically with other cytokines to enhance IFN-γ production, as it has previously been shown that TCR-stimulated CD8+ T cells produce more IFN-γ in the presence of IL-7 and IL-21 compared with IL-7 alone after 7 days of culture.43

When memory CD8+ T cells were cultured with IL-7, it was found that IL-7 did not enhance CD107a/b expression (Fig. 4c). While there is limited information regarding the effects of IL-7 on degranulation, IL-7 can enhance perforin expression,44 which may be coupled with degranulation. It is possible that enhanced degranulation was not observed because although IL-7 upregulates perforin, longer culture times may be required to lead to degranulation.

It has been noted that a population of CD45RA+CCR7 (TEMRA) cells emerged after 6 days of culture and exhibited significant proliferative and IFN-γ responses to antigen (Figs 2b and 3b). This is somewhat surprising given that the initial sorted population was CD45RA with a high degree of purity (99·10 ± 0·59%). Collectively, the cell-culture phenotype changed from 71·93 ± 8·85% TCM and 27·7 ± 8·57% TEM on day 0 to 38·55 ± 8·09% TCM, 38·18 ± 15·75% TEM and 5·32 ± 4·98% TEMRA after 6 days of culture in media alone (data not shown). Culture with antigen and IL-7 resulted in a similar subset distribution. It therefore appears that a subset of memory CD8+ T cells has the ability to differentiate into effector CD8+ T cells.

To determine if CD8CD45RA− CD127+ T-cell responses are impaired in HIV infection, proliferation, IFN-γ production and CD107a/b expression in response to antigen were evaluated. Antigen stimulation did not enhance the proliferation of CD8CD45RA− CD127+ T cells from HIV+ individuals (Fig. 5a), indicating the impairment of memory CD8+ T-cell responses in HIV infection. CD8+ T cells can proliferate during acute HIV infection, but lose this function as disease progresses.45,46

Several reports suggest general differences in the functional capacities of CD8+ T cells of HIV individuals compared with HIV+ individuals.6,47 IFN-γ production is another important function of activated memory CD8+ T cells, and, as demonstrated here, antigenic stimulation significantly enhanced IFN-γ production by CD8CD45RA− CD127+ T cells from HIV+ individuals; however, the extent of IFN-γ production was considerably less than in CD8CD45RA− CD127+ T cells from HIV individuals (Fig. 5b). Although the extent of impairment of non-HIV-specific CD8+ T-cell responses in untreated HIV+ individuals has not been fully investigated, the observed decrease in IFN-γ production in response to antigen indicates a degree of functional impairment. In a previous study, highly active anti-retroviral therapy (HAART)-treated HIV+ individuals had an increased proportion of IFN-γ-producing CD8+ T cells when pulsed with a CEF peptide pool,48 suggesting that the loss of CD8+ T-cell function can be reversed to some extent with effective treatment. CD8+ T-cell dysfunction has also been shown to be reversed, and HIV-specific CD8CD127hi T cells can become functional, long-lived memory CD8+ T cells if the viral load is suppressed early during infection with ART.49 The proportion of polyfunctional CD8+ T cells in HIV+ individuals can also increase with prolonged, successful ART.50 The lack of or limited response to antigen, as measured by proliferation (Fig. 5a) and IFN-γ production (Fig. 5b) in these experiments, suggests a functional impairment in non-HIV-specific CD8CD45RA− CD127+ T cells in untreated HIV+ individuals.

Previous studies have demonstrated impaired IL-7 activity on T cells during HIV infection; however, these studies included the investigation of unsorted T-cell populations that included low proportions of CD127+ cells, a hallmark of HIV infection.30,34,51 By isolating CD8CD45RA− CD127+ T cells, IL-7 activity could be directly compared between HIV and HIV+ individuals. IL-7 did not enhance antigen-mediated proliferation of CD8CD45RA− CD127+ T cells from HIV+ individuals (Fig. 5a), whereas in HIV individuals, proliferation was significantly enhanced by IL-7 (Fig. 2c). This is the first demonstration of impaired IL-7 activity in memory CD8+ T cells in HIV infection that cannot be attributed to a decreased proportion of CD127+ T cells, and this could be a reason for the loss of CD8+ T-cell function. This impairment of IL-7 activity may potentially be overcome with effective treatment, as recent clinical trials have shown that using IL-7 treatment in addition to HAART can increase the number of TN and TCM CD8+ T cells, and these cells can respond to antigenic stimulation.52 These results further our understanding of memory CD8+ T-cell responses in health and in HIV disease, and are relevant to current studies using IL-7 as a therapeutic agent.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

This research was supported by grants from the Ontario HIV Treatment Network (OHTN) Grant # ROGB131, the Canadian Institutes of Health Research Grant # HOP84649 and the Canadian Foundation for AIDS Research Grant # 019014. A.M.C. is a recipient of an OHTN fellowship and J.B.A. is an OHTN Career Scientist. A.M.O. designed and carried out experiments and wrote the paper. A.M.C. contributed to the initial project design, the data analysis, and helped write the paper. J.B.A. oversaw all aspects of the project and manuscript preparation.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
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