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Antigen load and viral sequence diversification determine the functional profile of HIV-1-specific CD8+ T cells.

Streeck H, Brumme ZL, Anastario M, Cohen KW, Jolin JS, Meier A, Brumme CJ, Rosenberg ES, Alter G, Allen TM, Walker BD, Altfeld M - PLoS Med. (2008)

Bottom Line: Virus-specific CD8(+) T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence.This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes.Monofunctionality increased in CD8(+) T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%-72%) to 76% (56%-95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%-75%) to 56% (42%-70%) (SD of the effect size 0.18) (p < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Partners AIDS Research Center, Infectious Disease Unit, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Virus-specific CD8(+) T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence. It has been suggested that virus-specific CD8(+) T cells with a "polyfunctional" profile, defined by the capacity to secrete multiple cytokines or chemokines, are most competent in controlling viral replication in chronic HIV-1 infection. We used HIV-1 infection as a model of chronic persistent viral infection to investigate the process of exhaustion and dysfunction of virus-specific CD8(+) T cell responses on the single-epitope level over time, starting in primary HIV-1 infection.

Methods and findings: We longitudinally analyzed the polyfunctional epitope-specific CD8(+) T cell responses of 18 patients during primary HIV-1 infection before and after therapy initiation or sequence variation in the targeted epitope. Epitope-specific CD8(+) T cells responded with multiple effector functions to antigenic stimulation during primary HIV-1 infection, but lost their polyfunctional capacity in response to antigen and up-regulated programmed death 1 (PD-1) expression with persistent viremic infection. This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes. Monofunctionality increased in CD8(+) T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%-72%) to 76% (56%-95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%-75%) to 56% (42%-70%) (SD of the effect size 0.18) (p < 0.05).

Conclusion: These data suggest that persistence of antigen can be the cause, rather than the consequence, of the functional impairment of virus-specific T cell responses observed during chronic HIV-1 infection, and underscore the importance of evaluating autologous viral sequences in studies aimed at investigating the relationship between virus-specific immunity and associated pathogenesis.

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Related in: MedlinePlus

Multiparameter Flow Cytometric Analysis of the Impact of Antiretroviral Therapy Initiation on the Functional Response Pattern of Antigen-Specific CD8+ T Cells(A) Gating scheme for identification of multifunctional CD8+ T cell responses. Single function gates were set based on negative control (unstimulated) samples and were placed consistently across samples. Initial gating was on the lymphocyte population. Doublets were removed in a forward scatter width (FSC-W) versus height (FSC-H) plot and live/dead discrimination was done subsequently with exclusion of high blue viability dye expression (UV). Sequentially CD3+ and CD8+ events were defined and gates for each respective function using combinations that provided optimal separation were made. After the gates for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 32 (25) response patterns when testing five functions. Shown here are the responses of patient Ac-160 to the HLA-B27-restricted epitope KK10 in p24 Gag. Data are reported after background correction.(B) Representative multifunctional composition of epitope-specific CD8+ T cell responses in an untreated patient identified during primary HIV-1 infection (Ac-177). Epitope-specific CD8+ T cell responses against four HIV-1 epitopes were studied during primary (early) and chronic (late) HIV-1 infection in this patient. All 32 possible combinations of the five antigen-specific functions studied for each epitope are shown on the x-axis and the contribution of each epitope-specific CD8+ T cell population exhibiting the respective combination of functions to the total epitope-specific response are indicated as bars. Responses are grouped and color-coded according to the number of functions (1 = yellow, 2 = cyan, 3 = green, 4 = blue, 5 = red). Dark grey bars show the results from the early sample; light grey bars show the results from the later samples. The data are summarized by pie charts in which each slice of the pie represents the fraction of the total epitope-specific response that consists of CD8+ T cells with the respective number of functions. Pie charts on the left represent the fraction of the respective epitope-specific CD8+ T cell responses during primary HIV-1 infection (early), while pie charts on the right represent the later time point (late).(C) Representative multifunctional composition of epitope-specific CD8+ T cell responses of a patient, who received HAART during primary HIV-1 infection (AC-85). Strategy of analysis and figure layout is same as described for (B). The functionality of epitope-specific CD8+ T cell responses against the HLA-B8-restricted epitope FL8 in Nef and HLA-A2-restricted epitope YI9 in RT remained stable or even slightly increased from a time point before (early) to a time point after (late) antiretroviral treatment initiation.
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pmed-0050100-g001: Multiparameter Flow Cytometric Analysis of the Impact of Antiretroviral Therapy Initiation on the Functional Response Pattern of Antigen-Specific CD8+ T Cells(A) Gating scheme for identification of multifunctional CD8+ T cell responses. Single function gates were set based on negative control (unstimulated) samples and were placed consistently across samples. Initial gating was on the lymphocyte population. Doublets were removed in a forward scatter width (FSC-W) versus height (FSC-H) plot and live/dead discrimination was done subsequently with exclusion of high blue viability dye expression (UV). Sequentially CD3+ and CD8+ events were defined and gates for each respective function using combinations that provided optimal separation were made. After the gates for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 32 (25) response patterns when testing five functions. Shown here are the responses of patient Ac-160 to the HLA-B27-restricted epitope KK10 in p24 Gag. Data are reported after background correction.(B) Representative multifunctional composition of epitope-specific CD8+ T cell responses in an untreated patient identified during primary HIV-1 infection (Ac-177). Epitope-specific CD8+ T cell responses against four HIV-1 epitopes were studied during primary (early) and chronic (late) HIV-1 infection in this patient. All 32 possible combinations of the five antigen-specific functions studied for each epitope are shown on the x-axis and the contribution of each epitope-specific CD8+ T cell population exhibiting the respective combination of functions to the total epitope-specific response are indicated as bars. Responses are grouped and color-coded according to the number of functions (1 = yellow, 2 = cyan, 3 = green, 4 = blue, 5 = red). Dark grey bars show the results from the early sample; light grey bars show the results from the later samples. The data are summarized by pie charts in which each slice of the pie represents the fraction of the total epitope-specific response that consists of CD8+ T cells with the respective number of functions. Pie charts on the left represent the fraction of the respective epitope-specific CD8+ T cell responses during primary HIV-1 infection (early), while pie charts on the right represent the later time point (late).(C) Representative multifunctional composition of epitope-specific CD8+ T cell responses of a patient, who received HAART during primary HIV-1 infection (AC-85). Strategy of analysis and figure layout is same as described for (B). The functionality of epitope-specific CD8+ T cell responses against the HLA-B8-restricted epitope FL8 in Nef and HLA-A2-restricted epitope YI9 in RT remained stable or even slightly increased from a time point before (early) to a time point after (late) antiretroviral treatment initiation.

Mentions: Cryopreserved PBMCs of the 2 mo and following time point used for ELISPOT assays (as described above) were thawed, resuspended to 1–2 × 106 cells/ml in R10 media (RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, 5.5 ml of HEPES buffer; pH 7.2 ± 0.2), and rested for 1–2 h at 37 °C; 5% CO2. PBMCs were then examined for viability by Trypan blue exclusion (typically 80%–90% viable) and adjusted to 1 × 106 cells/ml. Co-stimulatory antibodies (CD28 and 49d, 1 μg/ml; BD Biosciences) and CD107a-PE-Cy5 (BD Biosciences) were added and the cells aliquoted at 1 ml to each tube containing 2 μg/ml of each peptide. An unstimulated (R10 only) and a positive control (2 μl of phorbol-12-myristate-13-acetate 1mg/ml and 1 μl of ionomycin 1 mg/ml [AG Scientific]) were included in each assay. Cells were incubated for 30 min at 37 °C, 5% CO2, and monensin (Golgistop, 0.7 μl/ml; BD Biosciences) and brefeldin A (10 μg/ml; Sigma-Aldrich) were then added. Following a total incubation of 6 h, the cells were washed with PBS and stained for intracellular amine groups in order to discriminate between live and dead cells (blue viability dye, Invitrogen). Cells were washed again and then stained with anti-CD3-Pacific blue (BD Biosciences), anti-CD8-APC-Cy7 antibodies (BD Biosciences). Cells were then fixed in 1% paraformaldehyde (Fix Perm A), washed with PBS, and then permeabilized (Fix Perm B solution) (Caltag Laboratories). Cells were intracellularly stained using a panel of Il-2-FITC (BD Biosciences), IFN-γ-PE-Cy7 (BD Biosciences), TNF-α-Alexa700 (BD Biosciences), and MIP-1β-PE (BD Biosciences). Between 150,000 and 500,000 events were collected per sample. All data were collected on a BD LSRII (BD Biosciences) flow cytometer and analyzed using FlowJo 8.3.3 software (TreeStar). Initial gating was on the lymphocyte population and then used a forward scatter width (FSC-W) versus height (FSC-H) plot to remove doublets (see Figure 1A). Subsequently, the events were gated through a side scatter (SSC) versus blue viability dye (UV) and sequentially gated on CD3+ and CD8+ events. Following identification of CD8+ T cells, a gate was made for each respective function using combinations that provided optimal separation. After the gates for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 32 response patterns when testing five functions. Data are reported after background correction and the percent of epitope-specific CD8+ T cell responses had to be at least >2-fold higher than background for individual cytokines or CD107a before using the Boolean gate platform to be considered as a positive response.


Antigen load and viral sequence diversification determine the functional profile of HIV-1-specific CD8+ T cells.

Streeck H, Brumme ZL, Anastario M, Cohen KW, Jolin JS, Meier A, Brumme CJ, Rosenberg ES, Alter G, Allen TM, Walker BD, Altfeld M - PLoS Med. (2008)

Multiparameter Flow Cytometric Analysis of the Impact of Antiretroviral Therapy Initiation on the Functional Response Pattern of Antigen-Specific CD8+ T Cells(A) Gating scheme for identification of multifunctional CD8+ T cell responses. Single function gates were set based on negative control (unstimulated) samples and were placed consistently across samples. Initial gating was on the lymphocyte population. Doublets were removed in a forward scatter width (FSC-W) versus height (FSC-H) plot and live/dead discrimination was done subsequently with exclusion of high blue viability dye expression (UV). Sequentially CD3+ and CD8+ events were defined and gates for each respective function using combinations that provided optimal separation were made. After the gates for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 32 (25) response patterns when testing five functions. Shown here are the responses of patient Ac-160 to the HLA-B27-restricted epitope KK10 in p24 Gag. Data are reported after background correction.(B) Representative multifunctional composition of epitope-specific CD8+ T cell responses in an untreated patient identified during primary HIV-1 infection (Ac-177). Epitope-specific CD8+ T cell responses against four HIV-1 epitopes were studied during primary (early) and chronic (late) HIV-1 infection in this patient. All 32 possible combinations of the five antigen-specific functions studied for each epitope are shown on the x-axis and the contribution of each epitope-specific CD8+ T cell population exhibiting the respective combination of functions to the total epitope-specific response are indicated as bars. Responses are grouped and color-coded according to the number of functions (1 = yellow, 2 = cyan, 3 = green, 4 = blue, 5 = red). Dark grey bars show the results from the early sample; light grey bars show the results from the later samples. The data are summarized by pie charts in which each slice of the pie represents the fraction of the total epitope-specific response that consists of CD8+ T cells with the respective number of functions. Pie charts on the left represent the fraction of the respective epitope-specific CD8+ T cell responses during primary HIV-1 infection (early), while pie charts on the right represent the later time point (late).(C) Representative multifunctional composition of epitope-specific CD8+ T cell responses of a patient, who received HAART during primary HIV-1 infection (AC-85). Strategy of analysis and figure layout is same as described for (B). The functionality of epitope-specific CD8+ T cell responses against the HLA-B8-restricted epitope FL8 in Nef and HLA-A2-restricted epitope YI9 in RT remained stable or even slightly increased from a time point before (early) to a time point after (late) antiretroviral treatment initiation.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2365971&req=5

pmed-0050100-g001: Multiparameter Flow Cytometric Analysis of the Impact of Antiretroviral Therapy Initiation on the Functional Response Pattern of Antigen-Specific CD8+ T Cells(A) Gating scheme for identification of multifunctional CD8+ T cell responses. Single function gates were set based on negative control (unstimulated) samples and were placed consistently across samples. Initial gating was on the lymphocyte population. Doublets were removed in a forward scatter width (FSC-W) versus height (FSC-H) plot and live/dead discrimination was done subsequently with exclusion of high blue viability dye expression (UV). Sequentially CD3+ and CD8+ events were defined and gates for each respective function using combinations that provided optimal separation were made. After the gates for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 32 (25) response patterns when testing five functions. Shown here are the responses of patient Ac-160 to the HLA-B27-restricted epitope KK10 in p24 Gag. Data are reported after background correction.(B) Representative multifunctional composition of epitope-specific CD8+ T cell responses in an untreated patient identified during primary HIV-1 infection (Ac-177). Epitope-specific CD8+ T cell responses against four HIV-1 epitopes were studied during primary (early) and chronic (late) HIV-1 infection in this patient. All 32 possible combinations of the five antigen-specific functions studied for each epitope are shown on the x-axis and the contribution of each epitope-specific CD8+ T cell population exhibiting the respective combination of functions to the total epitope-specific response are indicated as bars. Responses are grouped and color-coded according to the number of functions (1 = yellow, 2 = cyan, 3 = green, 4 = blue, 5 = red). Dark grey bars show the results from the early sample; light grey bars show the results from the later samples. The data are summarized by pie charts in which each slice of the pie represents the fraction of the total epitope-specific response that consists of CD8+ T cells with the respective number of functions. Pie charts on the left represent the fraction of the respective epitope-specific CD8+ T cell responses during primary HIV-1 infection (early), while pie charts on the right represent the later time point (late).(C) Representative multifunctional composition of epitope-specific CD8+ T cell responses of a patient, who received HAART during primary HIV-1 infection (AC-85). Strategy of analysis and figure layout is same as described for (B). The functionality of epitope-specific CD8+ T cell responses against the HLA-B8-restricted epitope FL8 in Nef and HLA-A2-restricted epitope YI9 in RT remained stable or even slightly increased from a time point before (early) to a time point after (late) antiretroviral treatment initiation.
Mentions: Cryopreserved PBMCs of the 2 mo and following time point used for ELISPOT assays (as described above) were thawed, resuspended to 1–2 × 106 cells/ml in R10 media (RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, 5.5 ml of HEPES buffer; pH 7.2 ± 0.2), and rested for 1–2 h at 37 °C; 5% CO2. PBMCs were then examined for viability by Trypan blue exclusion (typically 80%–90% viable) and adjusted to 1 × 106 cells/ml. Co-stimulatory antibodies (CD28 and 49d, 1 μg/ml; BD Biosciences) and CD107a-PE-Cy5 (BD Biosciences) were added and the cells aliquoted at 1 ml to each tube containing 2 μg/ml of each peptide. An unstimulated (R10 only) and a positive control (2 μl of phorbol-12-myristate-13-acetate 1mg/ml and 1 μl of ionomycin 1 mg/ml [AG Scientific]) were included in each assay. Cells were incubated for 30 min at 37 °C, 5% CO2, and monensin (Golgistop, 0.7 μl/ml; BD Biosciences) and brefeldin A (10 μg/ml; Sigma-Aldrich) were then added. Following a total incubation of 6 h, the cells were washed with PBS and stained for intracellular amine groups in order to discriminate between live and dead cells (blue viability dye, Invitrogen). Cells were washed again and then stained with anti-CD3-Pacific blue (BD Biosciences), anti-CD8-APC-Cy7 antibodies (BD Biosciences). Cells were then fixed in 1% paraformaldehyde (Fix Perm A), washed with PBS, and then permeabilized (Fix Perm B solution) (Caltag Laboratories). Cells were intracellularly stained using a panel of Il-2-FITC (BD Biosciences), IFN-γ-PE-Cy7 (BD Biosciences), TNF-α-Alexa700 (BD Biosciences), and MIP-1β-PE (BD Biosciences). Between 150,000 and 500,000 events were collected per sample. All data were collected on a BD LSRII (BD Biosciences) flow cytometer and analyzed using FlowJo 8.3.3 software (TreeStar). Initial gating was on the lymphocyte population and then used a forward scatter width (FSC-W) versus height (FSC-H) plot to remove doublets (see Figure 1A). Subsequently, the events were gated through a side scatter (SSC) versus blue viability dye (UV) and sequentially gated on CD3+ and CD8+ events. Following identification of CD8+ T cells, a gate was made for each respective function using combinations that provided optimal separation. After the gates for each function were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 32 response patterns when testing five functions. Data are reported after background correction and the percent of epitope-specific CD8+ T cell responses had to be at least >2-fold higher than background for individual cytokines or CD107a before using the Boolean gate platform to be considered as a positive response.

Bottom Line: Virus-specific CD8(+) T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence.This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes.Monofunctionality increased in CD8(+) T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%-72%) to 76% (56%-95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%-75%) to 56% (42%-70%) (SD of the effect size 0.18) (p < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Partners AIDS Research Center, Infectious Disease Unit, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Virus-specific CD8(+) T lymphocytes play a key role in the initial reduction of peak viremia during acute viral infections, but display signs of increasing dysfunction and exhaustion under conditions of chronic antigen persistence. It has been suggested that virus-specific CD8(+) T cells with a "polyfunctional" profile, defined by the capacity to secrete multiple cytokines or chemokines, are most competent in controlling viral replication in chronic HIV-1 infection. We used HIV-1 infection as a model of chronic persistent viral infection to investigate the process of exhaustion and dysfunction of virus-specific CD8(+) T cell responses on the single-epitope level over time, starting in primary HIV-1 infection.

Methods and findings: We longitudinally analyzed the polyfunctional epitope-specific CD8(+) T cell responses of 18 patients during primary HIV-1 infection before and after therapy initiation or sequence variation in the targeted epitope. Epitope-specific CD8(+) T cells responded with multiple effector functions to antigenic stimulation during primary HIV-1 infection, but lost their polyfunctional capacity in response to antigen and up-regulated programmed death 1 (PD-1) expression with persistent viremic infection. This exhausted phenotype significantly decreased upon removal of stimulation by antigen, either in response to antiretroviral therapy or by reduction of epitope-specific antigen load in the presence of ongoing viral replication, as a consequence of in vivo selection of cytotoxic T lymphocyte escape mutations in the respective epitopes. Monofunctionality increased in CD8(+) T cell responses directed against conserved epitopes from 49% (95% confidence interval 27%-72%) to 76% (56%-95%) (standard deviation [SD] of the effect size 0.71), while monofunctionality remained stable or slightly decreased for responses directed against escaped epitopes from 61% (47%-75%) to 56% (42%-70%) (SD of the effect size 0.18) (p < 0.05).

Conclusion: These data suggest that persistence of antigen can be the cause, rather than the consequence, of the functional impairment of virus-specific T cell responses observed during chronic HIV-1 infection, and underscore the importance of evaluating autologous viral sequences in studies aimed at investigating the relationship between virus-specific immunity and associated pathogenesis.

Show MeSH
Related in: MedlinePlus