Longevity and composition of cellular immune responses following experimental Plasmodium falciparum malaria infection in humans

Abstract

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only 'adaptive' but also 'innate' lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO(+) CD62L(-) effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ(+)IL-2(+)) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field.

Figure 1. Flowchart of Experimental Human Malaria Infection study.

Black and white mosquito symbols indicate exposure to infected mosquito bites and uninfected mosquito bites, respectively. Development of patent blood-stage parasitemia following the first three inoculations was prevented by prophylactic chloroquine treatment, indicated by grey shading. Arrow heads indicate time points of immunological assessment: prior to immunization (I-1), prior to patent challenge (C-1), during expected blood-stage infection (C+9), two weeks after treatment (day C+35), 4.5 months post-challenge (day C+140) and again 1.1 year post-challenge (day C+400).

Figure 2. Induction and persistence of IFNγ responses to PfRBC and PfSpz during experimental malaria infection.

PBMC were isolated from volunteers prior to inclusion (day I-1), immediately prior to patent challenge (day C-1), during expected blood-stage malaria infection (day C+9), two weeks after treatment (day C+35), 4.5 months post-challenge (day C+140) and again 1.1 year post-challenge (day C+400). Note that Group A, but not Group B volunteers were exposed thrice to immunizing sub-patent infections between day I-1 and C-1 ( Figure 1 ). PBMC of volunteers of Group A (A+C) and Group B (B+D) were stimulated in vitro for 24 hours with PfRBC (A+B) or PfSpz (C+D) or their respective uninfected red blood cells (uRBC) or salivary glands from uninfected mosquitoes (MSG) controls, then stained for intracellular IFNγ and analyzed by flow cytometry. Shown are the percentage of total lymphocytes staining positive for IFNγ at each time point. Background responses were subtracted from the responses to parasite stimuli for every individual volunteer at every individual time point. Symbols represents responses by individual Group A volunteers (n = 10) and Group B volunteers (n = 5) for whom sufficient cells were available. Horizontal lines represent group medians. Median background values for uRBC were 0.01% [0.01–0.03] (median [IQR]) on I-1 up to C+35 and 0.03% [0.01–0.16] on C+140 and C+400. Background values for MSG were 0.02% [0.01–0.02] on I-1 up to C+35 and 0.07% [0.03–0.25] on C+140 and C+400.

Figure 3. Contribution of innate, semi-innate and adaptive lymphocyte subsets to the total IFNγ⁺ response to PfRBC.

PBMC isolated from Group A volunteers at the respective study time points were stimulated with PfRBC or uRBC and stained for intracellular IFNγ and surface expression of CD3, γδT and CD56 (gating strategy shown in Figure S1.A). Pie charts show the relative contributions of αβT cells (CD3⁺γδ^-CD56^-), γδT cells (CD3⁺γδ⁺CD56^-), NK cells (CD3^-γδ^-CD56⁺), NKT cells (CD3⁺γδ^-CD56⁺), ‘γδNKT’ cells (CD3⁺γδ⁺CD56⁺) and other lymphocytes to the total number of IFNγ⁺ cells responding to PfRBC (corrected for uRBC background). Shown are median values for ten Group A volunteers; pie chart surface areas directly correlate with the magnitude of (total) IFNγ+ responses. At time point I-1 the median [IQR] contribution of γδT cells, γδNKT cells, αβT cells & NK cells to total IFNγ responses was 63% [45–74], 11% [6.4–15], 15% [5.1–37] & 1.9% [0.3–5.6], respectively; at C+35 57%[ 47–59], 6.7% [4.0–8.9], 22% [17–28] and 4.1% [3.0–7.1] and C+400 35% [29–44], 11% [8.0–16], 25% [20–28] & 17% [12–25].

Figure 4. Contribution of EM and CM cells to the total IFNγ response to PfRBC and PfSpz.

PBMC isolated from volunteers at various study time points were stimulated in vitro for 24 hours with PfRBC (A+B) or PfSpz (C) and stained for the memory marker CD45RO and the homing marker CD62L. Bars show the contributions of effector memory (EM, CD45RO⁺CD62L^-), central memory (CM, CD45RO⁺CD62L⁺) and naive lymphocytes (CD45RO^-) to the total percentage of IFNγ-producing cells over time. Height of bars represents median values for Group A (A+C) and Group B (B) volunteers for the different cell subsets. Donors with insufficient numbers of IFNγ responding cells to assess the relative contribution of cell subsets were excluded from this composition analysis. Numbers below the bars represent the number of donors included per time point. ND – not done: insufficient numbers to reliably assess group medians; this was similarly the case for anti-PfSpz responses in group B. Since only donors with sufficient numbers of responding cells to assess the relative contribution of lymphocyte subsets are represented, these distributions may appear biased towards patterns in relatively stronger responders.

Figure 5. Immunological memory carriage by the γδT compartment vs other PBMC.

(A) Cryopreserved PBMC isolated from volunteers at inclusion (I) or 35 or 140 days post-challenge (C), were thawed and separated by magnetic beads into γδT⁺ lymphocytes (white) and remaining γδT^- PBMC (shades of grey, e.g. αβT cells, NK cells, B cells, monocytes). Following autologous re-combination at original ratios, PBMC were stimulated, stained and measured as for Figure 3 . (B) Shown are percentages of total lymphocytes staining IFNγ⁺ following incubation with PfRBC (corrected for uRBC background). Data represent median+IQR of seven volunteers from whom sufficient cells were available for the assay.

Figure 6. Uni- and polyfunctional EM T cell responses to PfRBC and PfSpz one year post-infection.

Data represent percentage of effector memory (EM) cells producing either IFNγ alone, IFNγ and IL-2, or IL-2 alone, following 24 hours in vitro stimulation with (A) PfRBC or (B) PfSpz at 400 days after challenge (C+400) for seven individual volunteers of Group A.