Comparative study of brain CD8+ T cells induced by sporozoites and those induced by blood-stage Plasmodium berghei ANKA involved in the development of cerebral malaria

Abstract

To obtain insight into the mechanisms that contribute to the pathogenesis of Plasmodium infections, we developed an improved rodent model that mimics human malaria closely by inducing cerebral malaria (CM) through sporozoite infection. We used this model to carry out a detailed study on isolated T cells recruited from the brains of mice during the development of CM. We compared several aspects of the immune response related to the experimental model of Plasmodium berghei ANKA infection induced by sporozoites in C57BL/6 mice and those related to a blood-stage infection. Our data show that in both models, oligoclonal TCRVbeta4(+), TCRVbeta6(+), TCRVbeta8.1(+), and TCRVbeta11(+) major histocompatibility complex class I-restricted CD8 T cells were present in the brains of CM(+) mice. These CD8(+) T cells display an activated phenotype, do not undergo apoptosis, secrete gamma interferon or tumor necrosis factor alpha, and are associated with the development of the neurological syndrome.

FIG. 1.

Comparison of CM in sporozoite- and PRBC-infected mice. (A) Survival curves for groups of five mice infected with different doses of sporozoites (SPZ) (1 × 10³, 5 × 10³, 1 × 10⁴, 5 × 10⁴, and 10⁵) and a group of eight mice infected with 10⁶ PRBC. (B) Parasitemia was estimated during the course of infection (days 3, 5, and 6) for all mice in the different groups by using Giemsa-stained, thick blood smears. (C) Student's t test was used to compare the levels of parasitemia on the day of CM manifestation for 14 CM⁺ and 6 CM⁻ mice infected with 50,000 sporozoites (SPZ) and for 13 CM⁺ and 4 CM⁻ mice infected with 10⁶ PRBC.

FIG. 2.

T-cell counts during CM in PBL, spleen, and LN. Total numbers of TCRαβ⁺, TCRγδ⁺, CD4⁺, and CD8⁺ among CD3⁺ cells are expressed per milliliter of blood for PBL and as the total number of cells for spleen and pooled peripheral LN taken when mice exhibited clinical signs of CM. Results for four CM⁺ PRBC-infected mice and eight CM⁺ sporozoite-infected mice are shown. For the two types of infection, eight uninfected mice were used as a control. Bar charts show the mean numbers of cells and standard deviation for two separate experiments.

FIG. 3.

Apoptosis occurs in peripheral CD4⁺ but not in CD8⁺ T-cell subpopulations. The percentage of early apoptotic cells (annexin V⁺ PI⁻) was determined among CD4⁺ and CD8⁺ T-cell subpopulations in the blood of uninfected controls (n = 5) and in mice infected with either sporozoites or PRBC and exhibiting clinical signs of CM (n = 5).

FIG. 4.

Selective accumulation of CD8⁺ T cells in the brain during CM. The analysis was done twice with T lymphocytes isolated from the pooled brains of nine control uninfected mice, three CM⁺ PRBC-infected mice, and five CM⁺ sporozoite-infected mice. □, control; ▪, CM.

FIG. 5.

Phenotype of CD8⁺ T lymphocytes isolated from the brains of CM⁺ mice. (A) Activation markers. Lymphoid cells isolated from brains of CM⁺ mice were doubly stained with anti-CD8 followed by anti-activation marker antibodies: CD25, CD44, CD62L, and CD69. Shown is a dot plot analysis indicating the activation markers expressed on naive cells from the LN of uninfected C57BL/6 mice (first row), CM⁺ mice infected with PRBC (second row), and CM⁺ mice infected with sporozoites (third row). (B) Adhesion molecules. The level of expression of the adhesion molecules on pooled lymphoid cells isolated from the brains of PRBC-infected CM⁺ mice after double staining with CD8 and ICAM-1 or LFA-1 (solid lines) was compared with that on naive T CD8⁺ cells isolated from the blood (dotted lines).

FIG. 6.

T cells in the brains of CM⁺ mice are not apoptotic. The percentage of early apoptotic cells (annexin V⁺ PI⁻) in pools of lymphoid cells isolated from the brains of five uninfected control mice and five CM⁺ mice infected with either sporozoites or PRBC is shown.

FIG. 7.

Cytokine spectrum of CD8⁺ T cells from CM⁺ mice. Lymphoid cells isolated from the brains of five CM⁺ mice were subjected to FACS analysis after intracellular staining for IFN-γ, TNF-α, IL-4, and IL-10. Cytokine production was analyzed on gated CD8-positive cells.

FIG. 8.

CD8⁺ T-cell repertoire during experimental CM. (A) Sorted CD8⁺ T lymphocytes isolated from the brains of five mice infected with sporozoites or PRBC were subjected to immunoscope analysis to determine the Vβ-Cβ repertoire. In this figure, only the profiles showing oligoclonal expansion are presented (Vβ4, Vβ6, Vβ8.1, and Vβ11); the other profiles had a Gaussian bell shape. (B) The percentage of TCR Vβ-positive cells was estimated by cytofluorometry of a pool of CD8 T cells isolated from the lymphoid compartments of five control or infected mice. ○, control; •, CM.

FIG. 9.

CM susceptibility of CD8-deficient mice. (A) Mortality due to CM in CD8^−/− (n = 5 for PRBC and n = 5 for sporozoite infection), β₂m^−/− (n = 10 for PRBC and n = 6 for sporozoites), and CD8 MAb-depleted (CD8 depl) (n = 5 for PRBC) mice after infection with 10⁶ PRBC or 50,000 sporozoites of P. berghei ANKA is shown. Survival curves for C57BL/6 mice (n = 5 for PRBC and n = 7 for sporozoites) are included as a reference for CM susceptibility. The shaded portion represents the time window of mortality from CM. (B) Parasitemia was determined on the day of death, whether it was early or late, for all the mice.

FIG. 10.

Susceptibility of H-2K^b, H-2D^b, H-2K^bD^b, and CD1 knockout mice to CM. (A) Mortality due to CM in K^b−/− (n = 10 for PRBC and n = 6 for sporozoites), D^b−/− (n = 9 for PRBC and n = 5 for sporozoites), CD1d^−/− (n = 10 for PRBC), and K^b−/−D^b−/− (n = 10 for PRBC and n = 6 for sporozoites) mice after infection with 10⁶ PRBC or 50,000 sporozoites of P. berghei ANKA is shown. Survival curves for C57BL/6 mice (n = 5 for PRBC and n = 6 for sporozoites) are included as a reference for CM susceptibility. The shaded portion represents the time window of mortality from CM. (B) Parasitemia was determined on the day of death for all the mice.

FIG. 11.

In vivo depletion of T cells bearing TCRVβ8.1 and TCRVβ8.2 segments. Survival of mice depleted for Vβ4 (n = 5), Vβ6 (n = 5), Vβ8.1 and Vβ8.2 (n = 5), and Vβ11 (n = 5) or for all of these Vβ subpopulations (n = 5) after infection with 10⁶ PRBC of P. berghei ANKA is shown. The C57BL/6 survival curve (n = 6) is included as a CM reference. The shaded part represents the time window of mortality from CM.