Flow cytometric detection of degranulation reveals phenotypic heterogeneity of degranulating CMV-specific CD8+ T lymphocytes in rhesus macaques

Abstract

Flow-cytometric conditions for detection of lysosomal-associated membrane proteins (LAMPs) on the surface of recently degranulated cells were optimized for rhesus macaques and used to investigate the functional properties of rhesus cytomegalovirus (rhCMV)-specific CD8+ T lymphocytes with regards to cytotoxicity and interferon (IFN)-gamma secretion in six asymptomatic CMV-seropositive rhesus macaques. Unlike humans, the rhesus macaque LAMP-1 protein CD107a underwent little or no endocytosis over a six to 18 h stimulation period. Following in vitro stimulation, rhCMV-specific CD8+ T lymphocytes were heterogeneous with regards to the composition of cells positive for CD107a and/or IFN-gamma, time to reach peak degranulation, and kinetics of IFN-gamma secretion relative to degranulation. Responder CD8+ T lymphocytes that underwent degranulation without IFN-gamma production (CD107a+IFN-gamma-) were predominantly composed of terminally differentiated effectors (CD28-CD45RA+). Moreover, they had significantly lower frequencies of effector memory (CD28-CD45RA-) cells compared to the IFN-gamma-secreting cells that did or did not undergo degranulation (CD107a+IFN-gamma+ or CD107a-IFN-gamma+). The perforin content of effector CD8+ T lymphocytes was significantly greater than that of effector memory CD8+ T lymphocytes in rhesus macaques, suggesting that they were more cytolytic. Our findings suggest that the composition of rhCMV-specific CD8+ T lymphocytes with regards to CD107a+IFN-gamma- responders may be an important determinant of their ability to control CMV replication.

Figure 1. Expression of LAMPs on T lymphocytes in rhesus macaques

PBMC were stimulated for six hours with (A) superantigen (SAg) and (B) rhCMV peptide IE1#83L in the presence of monensin and antibodies to CD107a (FITC), CD107b (PE) or CD63 (FITC) antibodies. Lymphocytes gated on CD8+ and CD4+ T cells are shown. Numbers denote frequency of the gated population expressing the corresponding LAMP.

Figure 2. Surface expression of CD107a is associated with decline in intracellular perforin

(A) Representative contour plot of SEB-stimulated CD8+ T lymphocytes at four hours post-stimulation. (B) Kinetics of surface CD107a expression and intracellular perforin following SEB stimulation. The percent maximal response at each time point is shown. Mean values of two experiments shown. Error bars show standard error of mean (SEM). (C) Inverse correlation between surface CD107a expression and intracellular perforin. Rho and P-Values calculated by the nonparametric Spearman Rank correlation test.

Figure 3. Optimal conditions for detecting degranulating CD8+ T lymphocytes in rhesus macaques

PBMC were stimulated with SEB and stained with antibodies to (A) CD107a (PE), (B) CD107b (FITC), or (C) both CD107a and CD107b, present throughout the stimulation period (“during”), or added at the end of the stimulation period (“post”). Gated CD8+ T lymphocytes are shown. (D) Comparison of surface LAMP expression when antibodies were added “during” or “post”. Data following 6 or 18 hours of SEB and rhCMV-CD8 peptide stimulation are shown. P-Values were calculated by the nonparametric Wilcoxon Signed Rank test.

Figure 4. Differential effects of monensin and brefeldin A on detection of CD107a, IFN-γ and TNF-α

PBMC from rhesus macaques were stimulated with SEB in the presence of monensin or brefeldin A for (A) six hours or (B) 18 hours and analyzed by four-color flow cytometry. Numbers denote frequency of gated CD8+ T lymphocytes. Comparison of (C) IFN-γ production and (D) CD107a expression of CD8+ T lymphocytes stimulated with SEB (left panels) or cognate rhCMV peptides (right panels) for six hours in the presence of brefeldin A (BFA) or monensin (Mon). P-Values were calculated by the nonparametric Wilcoxon Signed Rank test. (E - J) Effect of addition of different concentrations of monensin to 1 μg/ml of BFA on CD107a and IFN-γ detection in CD8+ T cells stimulated with SEB or rhCMV peptides in two macaques. Data after six hours of stimulation are shown. Concentrations of monensin ranging between 0.2 and 2 μM were used.

Figure 5. Heterogeneity in IFN-γ-secreting and cytolytic ability of CD8+ T lymphocytes

(A) Gating strategy showing responding subsets of SEB-stimulated CD8+ T lymphocytes. Three responding subsets shown; CD107a+IFN-γ− (I), CD107a−IFN-γ+ (II), and CD107a+IFN-γ+ (III). (B) Representative data on kinetic analysis of responding CD8+ T lymphocyte subsets, and (C) Differential kinetics of surface CD107a expression and IFN-γ production. (D) Composition of eleven rhCMV-specific CD8+ T lymphocytes responses in six rhesus macaques at the end of 18 hours of stimulation. All data obtained by four-color flow cytometry. Mean values are shown. Error bars represent SEM of two to four independent experiments performed one to four months apart.

Figure 6. Memory phenotype of responding CD8+ T lymphocyte subsets of rhCMV-specific CD8+ T lymphocytes analyzed by seven-color flow cytometry

(A) Delineation of memory populations of CD8+ T lymphocytes based on concurrent measurement of CD95, CD28 and CD45RA. Overlay plots of four subsets of CD95+ memory cells are shown. (B) Memory phenotype of CD107a+IFN-γ− (I), CD107a−IFN-γ+ (II), and CD107a+IFN-γ+ (III) functional of rhCMV-specific CD8+ T lymphocytes at the end of an 18-hour stimulation period. P-Values were determined by repeated measures ANOVA and the Bonferroni post-hoc test. The frequency of CM1 and CM2 cells were significantly lower (P <0.05) than the Eff and EM populations for all three responding subsets (not shown on graph). The flow cytometry panel consisted of CD3 PB, CD8 Alexa700, CD95 APC, CD28 FITC, CD45RA PerCP, CD107a PE, and IFN-γ PE-Cy7 antibodies.

Figure 7. Cytolytic granule content of effector (CD95+CD28-CD45RA+) and effector memory (CD95+CD28- 45RA-) CD8+ T lymphocytes in rhesus macaques

(A) Overlay histograms showing granzyme B (left) and perforin (right) content of different memory populations of CD8+ T lymphocytes. (B) Comparison of the frequency of granzyme B+ and perforin+ cells between effector (Eff) and effector memory (EM) CD8+ T lymphocytes. (C) Comparison of the median channel fluorescence (MCF) of granyme B and perforin content between effectors (Eff) and effector memory (EM) CD8+ T lymphocytes. P-Values were calculated by the non-parametric Wilcoxon signed rank test. The eight-color flow cytometry panel consisted of CD3 APC-Cy7, CD4 PerCP, CD8 Alexa700, CD95 APC, CD28 ECD, CD45RA PE-Cy7, perforin FITC, and granzyme B PE antibodies.