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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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SIV-specific CD8+ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).A. The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). B. ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4+ T cell lines were used as targets. CD8+ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.
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ppat-1003195-g001: SIV-specific CD8+ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).A. The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). B. ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4+ T cell lines were used as targets. CD8+ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.

Mentions: To measure CD8+ T-cell cytotoxic capacity, we adapted use of these CD4+ T-cell lines to our assay previously used in humans [12]–[14]. Briefly, autologous macaque CD4+ T-cell lines were infected with SIV and mixed with PBMC for 6 days to stimulate effectors. In prior work in humans, we observed that this stimulation provided optimal loading with cytolytic molecules and cytotoxicity. CD8+ T cells were then negatively isolated and mixed with a second aliquot of surface labeled infected or uninfected autologous CD4+ T-cell lines. This mixing is performed in the presence of a cell permeable GrB substrate that fluoresces upon delivery of active GrB to target cells. After 1 hour, GrB delivery was measured by flow cytometry (Figure 1). The remaining cells were fixed, permeablized, and stained for CD4 and intracellular SIV p27 antigen. Cytotoxicity was measured as the fraction of targets to which active GrB was delivered, and the fraction of HIV infected targets that were eliminated (ICE) in a 1-hour period.


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)

SIV-specific CD8+ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).A. The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). B. ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4+ T cell lines were used as targets. CD8+ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585127&req=5

ppat-1003195-g001: SIV-specific CD8+ T cell cytotoxicity measured by granzyme B delivery or Infected CD4 Elimination (ICE).A. The top panels show granzyme B (GrB) target cell activity representative of a “high responder”. The bottom panels show GrB target cell activity representative of a “low responder”. Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Background GrB target cell activity measured in response to uninfected targets (left column) was subtracted from responses measured against infected targets (right column) to determine net GrB target cell activity (red values). B. ICE values calculated based on p27 expression (sum of the upper quadrants) as described in the Methods, are shown in red for the same “high responder” (78.8%, top row) and “low responder” (22.3%, bottom row) as shown in A. Quadrant values indicate percentages of gated targets. In all experiments, CD4+ T cell lines were used as targets. CD8+ T cells that had been stimulated with SIV-infected targets for 6 days were used as effectors. GrB target cell activity and ICE were calculated after 1 hour of incubation of effectors and plated at an E∶T ratio of 25∶1.
Mentions: To measure CD8+ T-cell cytotoxic capacity, we adapted use of these CD4+ T-cell lines to our assay previously used in humans [12]–[14]. Briefly, autologous macaque CD4+ T-cell lines were infected with SIV and mixed with PBMC for 6 days to stimulate effectors. In prior work in humans, we observed that this stimulation provided optimal loading with cytolytic molecules and cytotoxicity. CD8+ T cells were then negatively isolated and mixed with a second aliquot of surface labeled infected or uninfected autologous CD4+ T-cell lines. This mixing is performed in the presence of a cell permeable GrB substrate that fluoresces upon delivery of active GrB to target cells. After 1 hour, GrB delivery was measured by flow cytometry (Figure 1). The remaining cells were fixed, permeablized, and stained for CD4 and intracellular SIV p27 antigen. Cytotoxicity was measured as the fraction of targets to which active GrB was delivered, and the fraction of HIV infected targets that were eliminated (ICE) in a 1-hour period.

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