Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans

Abstract

Background: Immunity to malaria develops naturally in endemic regions, but the protective immune mechanisms are poorly understood. Many vaccination strategies aim to induce T cells against diverse pre-erythrocytic antigens, but correlates of protection in the field have been limited. The objective of this study was to investigate cell-mediated immune correlates of protection in natural malaria. Memory T cells reactive against thrombospondin-related adhesive protein (TRAP) and circumsporozoite (CS) protein, major vaccine candidate antigens, were measured, as were frequencies of CD4(+) CD25(high) T cells, which may suppress immunity, and CD56(+) NK cells and gammadelta T cells, which may be effectors or may modulate immunity.

Methodology and principal findings: 112 healthy volunteers living in rural Kenya were entered in the study. Memory T cells reactive against TRAP and CS were measured using a cultured IFNgamma ELISPOT approach, whilst CD4(+) CD25(high) T cells, CD56(+) NK cells, and gammadelta T cells were measured by flow cytometry. We found that T cell responses against TRAP were established early in life (<5 years) in contrast to CS, and cultured ELISPOT memory T cell responses did not correlate with ex-vivo IFNgamma ELISPOT effector responses. Data was examined for associations with risk of clinical malaria for a period of 300 days. Multivariate logistic analysis incorporating age and CS response showed that cultured memory T cell responses against TRAP were associated with a significantly reduced incidence of malaria (p = 0.028). This was not seen for CS responses. Higher numbers of CD4(+) CD25(high) T cells, potentially regulatory T cells, were associated with a significantly increased risk of clinical malaria (p = 0.039).

Conclusions: These data demonstrate a role for central memory T cells in natural malarial immunity and support current vaccination strategies aimed at inducing durable protective T cell responses against the TRAP antigen. They also suggest that CD4(+) CD25(high) T cells may negatively affect naturally acquired malarial immunity.

Figure 1. Relationship between ELISPOT responses and age.

Cultured ELISPOT responses against (a) TRAP and (b) CS for individuals were stratified according to age into 4 groups: 0–5, 5–10, 10–20, and over 20. Median, 25^th and 75^th quartile, 5^th and 95^th quartile and outlying points are given by box and whisker plots.

Figure 2. Correlations between ELISPOT responses.

The relationship was examined between (a) TRAP and CS responses both ex-vivo, and (b) TRAP and CS responses both cultured.

Figure 3. Intracellular IFNγ Staining following culture.

ICS for IFNγ was carried out on cells by restimulation with peptides, following 10 days of culture, and co-staining with CD4 or CD8. The mean percentages (±standard deviation) of IFNγ⁺ cells possessing CD4 or CD8 (with medium controls subtracted) are shown together with the mean CD4:CD8 ratio.

Figure 4. Characterisation of regulatory T cells and NK cells.

Ex-vivo PBMC were stained for surface CD4, CD25 and CD127, and intracellularly for FoxP3. Typical dot plots show (a) the distribution of CD25 determining CD25^high status, (b) the distribution of CD56 on CD3 negative (NK) cells determining CD56^dim and CD56^bright status, (c) that the majority of FoxP3⁺ cells are CD25^high, and (d) that the FoxP3⁺ cells are predominantly CD127⁻ . (e) A correlation was demonstrated between CD25^high and FoxP3 positivity.

Figure 5. Kaplan-Meier malaria-free survival plots.

Individuals were stratified into low, medium and high cultured ELISPOT responders (SFC/10⁶) to (a) TRAP and (b) CS and probability of remaining free of clinical malaria is plotted over the 300 day monitoring period. Co-variants incorporated into the analysis are age and antigen response to the other antigen.