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Cytotoxic capacity of SIV-specific CD8(+) T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques.

Mendoza D, Migueles SA, Rood JE, Peterson B, Johnson S, Doria-Rose N, Schneider D, Rakasz E, Trivett MT, Trubey CM, Coalter V, Hallahan CW, Watkins D, Franchini G, Lifson JD, Connors M - PLoS Pathog. (2013)

Bottom Line: Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date.In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed.These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans.

View Article: PubMed Central - PubMed

Affiliation: HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.

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PBMC from progressors can be expanded in vitro after treatment with phorbol esther and calcium ionophore.A. Three Mamu A*01+ rhesus macaques (894L, 887L, DBGR) were stimulated for 6 hours with phorbol-12-myristate-13-acetate (PMA, 400 ng/ml) and ionomycin (Io, 2 µM) on day 0, washed, re-suspended in medium containing 40 IU/mL of IL-2 and cultured for at least 30 days. Top medium was replaced with fresh IL-2-containing medium and counted every 24–48 hours. The controls were not treated with PMA/Io on day 0 but were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies during the first 6 days. B. PBMC from another Mamu A*01+ rhesus macaque were stimulated with the same concentrations of PMA/Io for 6 hours on day 0 and propagated in culture for an additional 18 days as described above. A pool containing 15-mers spanning the entire SIV Gag sequence was used to stimulate half of the cells on day 18. PBMC were stained on day 24 with the Mamu A01-restricted CTPYDINQM (CM9) tetramer to measure SIV-specific expansion.
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ppat-1003195-g005: PBMC from progressors can be expanded in vitro after treatment with phorbol esther and calcium ionophore.A. Three Mamu A*01+ rhesus macaques (894L, 887L, DBGR) were stimulated for 6 hours with phorbol-12-myristate-13-acetate (PMA, 400 ng/ml) and ionomycin (Io, 2 µM) on day 0, washed, re-suspended in medium containing 40 IU/mL of IL-2 and cultured for at least 30 days. Top medium was replaced with fresh IL-2-containing medium and counted every 24–48 hours. The controls were not treated with PMA/Io on day 0 but were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies during the first 6 days. B. PBMC from another Mamu A*01+ rhesus macaque were stimulated with the same concentrations of PMA/Io for 6 hours on day 0 and propagated in culture for an additional 18 days as described above. A pool containing 15-mers spanning the entire SIV Gag sequence was used to stimulate half of the cells on day 18. PBMC were stained on day 24 with the Mamu A01-restricted CTPYDINQM (CM9) tetramer to measure SIV-specific expansion.

Mentions: It remained unclear whether the cytotoxic capacity of SIV-specific CD8+ T-cells from progressors was permanently lost through exhaustion, deletion, or replicative senescence [28]–[31]. In prior work in humans, we did not observe increases in cytotoxic capacity of CD8+ T cells of progressors following stimulation with IL-2, IL-15, or stimulation through CD28 and the T-cell receptor [12]. However, we did observe that bypassing TCR stimulation by treatment with phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) followed by a period of rest can restore the proliferative and cytotoxic defects of HIV-specific CD8+ T cells in vitro. [12]. Therefore, we explored whether PBMC of progressor rhesus macaques could similarly be expanded by treatment with PMA/Io in vitro. PBMC from 3 additional Mamu A01+ progressors (894L, 887L and DBGR) were stimulated with either 400 ng/mL of PMA and 2 µM of ionomycin or anti-CD3 and anti-CD28 monoclonal antibodies (Figure 5A). In preliminary experiments, it had been determined that these concentrations of PMA/Io induced maximal expansion following 6-hour stimulation and that medium containing exogenous IL-2 was required to support prolonged in vitro propagation of PBMC (data not shown). Strikingly, the median peak expansion in the cultures treated with PMA/Io was 224-fold [range: 106–241], which occurred during the third week (Figure 5A). In contrast, the median expansion was only 3-fold [range: 3–12] in the anti-CD3/CD28-stimulated controls.


Cytotoxic capacity of SIV-specific CD8(+) T cells against primary autologous targets correlates with immune control in SIV-infected rhesus macaques.

Mendoza D, Migueles SA, Rood JE, Peterson B, Johnson S, Doria-Rose N, Schneider D, Rakasz E, Trivett MT, Trubey CM, Coalter V, Hallahan CW, Watkins D, Franchini G, Lifson JD, Connors M - PLoS Pathog. (2013)

PBMC from progressors can be expanded in vitro after treatment with phorbol esther and calcium ionophore.A. Three Mamu A*01+ rhesus macaques (894L, 887L, DBGR) were stimulated for 6 hours with phorbol-12-myristate-13-acetate (PMA, 400 ng/ml) and ionomycin (Io, 2 µM) on day 0, washed, re-suspended in medium containing 40 IU/mL of IL-2 and cultured for at least 30 days. Top medium was replaced with fresh IL-2-containing medium and counted every 24–48 hours. The controls were not treated with PMA/Io on day 0 but were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies during the first 6 days. B. PBMC from another Mamu A*01+ rhesus macaque were stimulated with the same concentrations of PMA/Io for 6 hours on day 0 and propagated in culture for an additional 18 days as described above. A pool containing 15-mers spanning the entire SIV Gag sequence was used to stimulate half of the cells on day 18. PBMC were stained on day 24 with the Mamu A01-restricted CTPYDINQM (CM9) tetramer to measure SIV-specific expansion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585127&req=5

ppat-1003195-g005: PBMC from progressors can be expanded in vitro after treatment with phorbol esther and calcium ionophore.A. Three Mamu A*01+ rhesus macaques (894L, 887L, DBGR) were stimulated for 6 hours with phorbol-12-myristate-13-acetate (PMA, 400 ng/ml) and ionomycin (Io, 2 µM) on day 0, washed, re-suspended in medium containing 40 IU/mL of IL-2 and cultured for at least 30 days. Top medium was replaced with fresh IL-2-containing medium and counted every 24–48 hours. The controls were not treated with PMA/Io on day 0 but were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies during the first 6 days. B. PBMC from another Mamu A*01+ rhesus macaque were stimulated with the same concentrations of PMA/Io for 6 hours on day 0 and propagated in culture for an additional 18 days as described above. A pool containing 15-mers spanning the entire SIV Gag sequence was used to stimulate half of the cells on day 18. PBMC were stained on day 24 with the Mamu A01-restricted CTPYDINQM (CM9) tetramer to measure SIV-specific expansion.
Mentions: It remained unclear whether the cytotoxic capacity of SIV-specific CD8+ T-cells from progressors was permanently lost through exhaustion, deletion, or replicative senescence [28]–[31]. In prior work in humans, we did not observe increases in cytotoxic capacity of CD8+ T cells of progressors following stimulation with IL-2, IL-15, or stimulation through CD28 and the T-cell receptor [12]. However, we did observe that bypassing TCR stimulation by treatment with phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) followed by a period of rest can restore the proliferative and cytotoxic defects of HIV-specific CD8+ T cells in vitro. [12]. Therefore, we explored whether PBMC of progressor rhesus macaques could similarly be expanded by treatment with PMA/Io in vitro. PBMC from 3 additional Mamu A01+ progressors (894L, 887L and DBGR) were stimulated with either 400 ng/mL of PMA and 2 µM of ionomycin or anti-CD3 and anti-CD28 monoclonal antibodies (Figure 5A). In preliminary experiments, it had been determined that these concentrations of PMA/Io induced maximal expansion following 6-hour stimulation and that medium containing exogenous IL-2 was required to support prolonged in vitro propagation of PBMC (data not shown). Strikingly, the median peak expansion in the cultures treated with PMA/Io was 224-fold [range: 106–241], which occurred during the third week (Figure 5A). In contrast, the median expansion was only 3-fold [range: 3–12] in the anti-CD3/CD28-stimulated controls.

Bottom Line: Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date.In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed.These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans.

View Article: PubMed Central - PubMed

Affiliation: HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Although the study of non-human primates has resulted in important advances for understanding HIV-specific immunity, a clear correlate of immune control over simian immunodeficiency virus (SIV) replication has not been found to date. In this study, CD8(+) T-cell cytotoxic capacity was examined to determine whether this function is a correlate of immune control in the rhesus macaque (RM) SIV infection model as has been suggested in chronic HIV infection. SIVmac251-infected human reverse transcriptase (hTERT)-transduced CD4(+) T-cell clone targets were co-incubated with autologous macaque effector cells to measure infected CD4(+) T-cell elimination (ICE). Twenty-three SIV-infected rhesus macaques with widely varying plasma viral RNA levels were evaluated in a blinded fashion. Nineteen of 23 subjects (83%) were correctly classified as long-term nonprogressor/elite controller (LTNP/EC), slow progressor, progressor or SIV-negative rhesus macaques based on measurements of ICE (weighted Kappa 0.75). LTNP/EC had higher median ICE than progressors (67.3% [22.0-91.7%] vs. 23.7% [0.0-58.0%], p = 0.002). In addition, significant correlations between ICE and viral load (r = -0.57, p = 0.01), and between granzyme B delivery and ICE (r = 0.89, p<0.001) were observed. Furthermore, the CD8(+) T cells of LTNP/EC exhibited higher per-cell cytotoxic capacity than those of progressors (p = 0.004). These findings support that greater lytic granule loading of virus-specific CD8(+) T cells and efficient delivery of active granzyme B to SIV-infected targets are associated with superior control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. Therefore, such measurements appear to represent a correlate of control of viral replication in chronic SIV infection and their role as predictors of immunologic control in the vaccine setting should be evaluated.

Show MeSH
Related in: MedlinePlus