Malaria protection in beta 2-microglobulin-deficient mice lacking major histocompatibility complex class I antigens: essential role of innate immunity, including gammadelta T cells

Abstract

It is still controversial whether malaria protection is mediated by conventional immunity associated with T and B cells or by innate immunity associated with extrathymic T cells and autoantibody-producing B cells. Given this situation, it is important to examine the mechanism of malaria protection in beta(2)-microglobulin-deficient (beta(2)m(-/-)) mice. These mice lack major histocompatibility complex class I and CD1d antigens, which results in the absence of CD8(+) T cells and natural killer T (NKT) cells. When C57BL/6 and beta(2)m(-/-) mice were injected with parasitized (Plasmodium yoelii 17XNL) erythrocytes, both survived from the infection and showed a similar level of parasitaemia. The major expanding T cells were NK1.1(-) alphabeta T-cell receptor(int) cells in both mice. The difference was a compensatory expansion of NK and gammadelta T cells in beta(2)m(-/-) mice, and an elimination experiment showed that these lymphocytes were critical for protection in these mice. These results suggest that malaria protection might be events of the innate immunity associated with multiple subsets with autoreactivity. CD8(+) T and NKT cells may be partially related to this protection.

Figure 1

Time-kinetic study after malarial infection. (a) Parasitaemia. (b) Number of lymphocytes yielded by the liver and spleen. B6 and β₂m(–/–) mice were infected with 10⁴ parasitized erythrocytes for the blood stage of malarial infection. At each point of time, four mice were used to produce the mean and one SD. *P < 0·05.

Figure 2

Phenotypic characterization of lymphocytes in immunofluorescence tests. (a) Two-colour staining for CD3 and IL-2Rβ. (b) Two-colour staining for γδTCR and NK1.1. Lymphocytes were isolated from the liver and spleen of B6 and β₂m(–/–) mice before and after malarial infection. Numbers in the figure represent the percentages of immunofluorescence-positive cells. Representative results from three experiments were depicted.

Figure 3

Variation in the number of NK cells, γδT cells and intermediate T cells in the liver during malarial infection. B6 and β₂m(–/–) were used. The mean and one SD were produced from four mice. *P < 0·05.

Figure 4

Experiments in which NK cells and γδT cells were eliminated in mice with malaria. (a) Parasitaemia. (b) Elimination of NK cells. (c) Elimination of γδT cells. NK cells were eliminated by anti-NK1.1 mAb, whereas γδT cells were eliminated by anti-γδTCR mAb. Representative results of three mice are depicted in each experiment. Some mice in which NK cells or γδT cells were eliminated died during malarial infection. Experiments (a) and (b) were two-colour immunofluorescence tests to detect NK cells and γδT cells.

Figure 5

Further characterization of the phenotype of γδT cells in mice with or without malaria. Three-colour staining of hepatic lymphocytes for IL-2Rβ, γδTCR and B220 (or CD5 or CD69) were conducted. Malaria-infected mice were used on day 21 after the infection. By gated analysis, the expressions of B220, CD5 and CD69 on γδT cells were estimated.

Figure 6

Usage of γδTCR by γδT cells identified by RT-PCR method. The signs of Vγ1, Vγ2, Vγ4, Vγ5 and Vγ6 were determined by RT–PCR method before and after malarial infection in B6 and β₂m(–/–) mice. The sign of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was a positive control.