Phenotypic profiling of CD8(+) T cells during Plasmodium vivax blood-stage infection

Abstract

Background: For a long time, the role of CD8(+) T cells in blood-stage malaria was not considered important because erythrocytes do not express major histocompatibility complex (MHC) class I proteins. While recent evidences suggest that CD8(+) T cells may play an important role during the erythrocytic phase of infection by eliminating parasites, CD8(+) T cells might also contribute to modulate the host response through production of regulatory cytokines. Thus, the role of CD8(+) T cells during blood-stage malaria is unclear. Here, we report the phenotypic profiling of CD8(+) T cells subsets from patients with uncomplicated symptomatic P. vivax malaria.

Methods: Blood samples were collected from 20 Plasmodium vivax-infected individuals and 12 healthy individuals. Immunophenotyping was conducted by flow cytometry. Plasma levels of IFN-γ, TNF-α and IL-10 were determined by ELISA/CBA. Unpaired t-test or Mann-Whitney test was used depending on the data distribution.

Results: P. vivax-infected subjects had lower percentages and absolute numbers of CD8(+)CD45RA(+) and CD8(+)CD45RO(+) T cells when compared to uninfected individuals (p ≤ 0.0002). A significantly lower absolute number of circulating CD8(+)CD45(+)CCR7(+) cells (p = 0.002) was observed in P. vivax-infected individuals indicating that infection reduces the number of central memory T cells. Cytokine expression was significantly reduced in the naïve T cells from infected individuals compared with negative controls, as shown by lower numbers of IFN-γ(+) (p = 0.001), TNF-α(+) (p < 0.0001) and IL-10(+) (p < 0.0001) CD8(+) T cells. Despite the reduction in the number of CD8(+) memory T cells producing IFN-γ (p < 0.0001), P. vivax-infected individuals demonstrated a significant increase in memory CD8(+)TNF-α(+) (p = 0.016) and CD8(+)IL-10(+) (p = 0.004) cells. Positive correlations were observed between absolute numbers of CD8(+)IL-10(+) and numbers of CD8(+)IFN-γ(+) (p < 0.001) and CD8(+)TNF-α(+) T cells (p ≤ 0.0001). Finally, an increase in the plasma levels of TNF-α (p = 0.017) and IL-10 (p = 0.006) and a decrease in the IFN-γ plasma level (p <0.0001) were observed in the P. vivax-infected individuals.

Conclusions: P. vivax infection reduces the numbers of different subsets of CD8(+) T cells, particularly the memory cells, during blood-stage of infection and enhances the number of CD8(+) memory T cells expressing IL-10, which positively correlates with the number of cells expressing TNF-α and IFN-γ.

Figure 1

Flow cytometric analysis of CD8 ⁺ naïve, double positive and memory T cells from malaria-naïve donors (n = 12) and P. vivax -infected donors (n = 17). A. CD8⁺CD45RA⁺. B. CD8⁺CD45RA⁺CD45RO⁺. C. CD8⁺CD45RO⁺. Results were expressed in absolute numbers (cells/mm³). Mann–Whitney test was used for comparison and the results were expressed as the median with interquartile range.

Figure 2

Flow cytometric analysis of central and effector memory of CD8 ⁺ T cells in malaria-naïve donors (n = 12) and P. vivax -infected donors (n = 17). A. Central memory (CD8⁺CD45RO⁺CCR7⁺/CD62L⁺). B. Effector memory (CD8⁺CD45RO⁺CCR7⁻/CD62L⁻). Mann–Whitney test was used for comparison and the results were expressed as the median with interquartile range. P values are indicated in the graphs.

Figure 3

Cytokine expression in CD8 ⁺ naïve, double positive and memory T cells. Expression of inflammatory (IFN-γ and TNF-α) and regulatory (IL-10) cytokines in malaria-naïve (n = 12) and P. vivax-infected donors (n = 17). The results are expressed in absolute numbers (cells/mm³). A. CD45RA⁺. B. CD45RA⁺CD45RO⁺. C. CD45RO⁺.

Figure 4

Correlation of peripheral blood CD8 ⁺ T cells expressing IL-10 vs . IFN-γ and IL-10 vs . TNF-α from P. vivax donors. A. CD45RA⁺. B. CD45RA⁺CD45RO⁺. C. CD45RO⁺. Results are expressed in absolute numbers (cells/mm³). Statistical significance was determined by Spearman correlation.

Figure 5

Ratio between pro- and anti-inflammatory cytokines produced by CD8 ⁺ T cells. Results represent the ratio between pro-inflammatory (IFN-γ or TNF-α) and anti-inflammatory (IL-10) cytokine production in malaria-naïve (n = 12) and P. vivax-infected donors (n = 17). A. naïve CD45RA⁺CD8⁺ T cells. B. double-positive CD45RA⁺CD45RO⁺ CD8⁺ T cells. C. memory CD45RO⁺CD8⁺ T cells. Mann–Whitney test was used for comparison and the result was expressed as the median with interquartile range.

Figure 6

Plasma levels of IFN-γ, TNF-α and IL-10. Levels of circulating IFN-γ and TNF-α were determined by ELISA, IL-10 was determined by cytometric bead array (CBA) in malaria-naïve donors (n = 12) and P. vivax-infected donors (n = 17). A. IFN-γ. B. TNF-α. C. IL-10. Mann–Whitney test was used for comparison and the result was expressed as the median with interquartile range. P values are indicated in the graphs.