Little progress has been made in elucidating the requirement for immune protection against malaria infection. Protection has not been achieved even with high titers of antibodies against known liver antigen [
33], blood stage antigen [
34,
35] or antigen specific CD8+ T cells produced with adenovirus immunization [
36]. In addition, human immunization with irradiated sporozoites has shown limited success unless sporozoites are injected intravenously [
37]. Conversely, immunization with irradiated sporozoites or live sporozoites under CQ cover induces protection [
29,
31]. In the current study, the infection/cure C57BL/6 mouse model was used to elucidate the immune protection conferred during malaria infection by characterizing the nature of CD8+ T cells. Recent studies showed that both CD4+ T cells and CD8+ T cells are necessary to mediate immunity to liver stage malaria parasites [
31,
32]. This was demonstrated by immunizing BALB/c mice with
Plasmodium yoelii iRBC under CQ cover and then treating with depleting antibodies against CD4+ T cells and CD8+ T cells. In this study, the infection/cure CD8+ T cells deficient (MHC I KO) mice resulted in only partial protection from lethal challenge, thus implicating CD8+ T cells in protection. Passive transfer with serum or cell alone also failed to confer 100% protection, thus indicating the cooperation of antigen specific cells and antibodies in granting protection. MHC II animals were also only partially protected. The most likely explanation is that the protective antibodies generated during infection/cure need CD4+ T cell help, which is lost in CD4+ T cell deficient animals. However, the role of CD4+ T cell in this model cannot be excluded is also to support the generation of protective CD8+ T cells or that they are directly involved in parasite killing. It will be interesting to perform transfer experiments to test the protective effect of serum from infection/cure MHC II animals enriched with CD4+ T cells from infection/cure wildtype animals. This will be important to discriminate between the roles of CD4+ T cells as pure ‘helper’ for antibodies production or more direct involved in anti-plasmodium activity. From this study and that by Belnoue
et al., data strongly suggest that both a humoral response and CD8+ T cells are required for immunity to liver stage
P. berghei parasites [
31,
32].
In an effort to further elucidate immune protection, CD8+ T cells in peripheral blood and splenocytes were characterized. The distinct populations of CD8+ T cells with phenotype KLRG1
high, CD27
low, CD44
high, CD62L
low were shown to be associated with protection. This population is absent in the intrahepatic lymphocytes. In the classical model of protection by immunization with irradiated sporozoites, the putative protective CD8+ T cells are found mostly in the liver [
18,
19] and produce IFNγ. In a recent study, Nganou-Makandop
et al.[
21] reported comparable levels of hepatic CD8+ T cells with a CD44
high, CD62L
low phenotype that produce IFNγ in response to PMA/Iono. In this study, mice were either immunized with radiation-attenuated sporozoites or infected with
P. berghei under CQ cover. In the current study, the CD8+ KLRG1
high, CD27
low, CD44
high, CD62L
low phenotype that was associated with protection was not present in the liver of immunized animals. A possible explanation of the apparent discrepancy is that more than one population of CD8+ T cells contributes to protection. One can speculate that the splenic CD8+ KLRG1
high, CD27
low, CD44
high, CD62L
low cells and the intrahepatic CD8+ CD44
high, CD62L
low cells are the same antigen specific population whose phenotype and organ-homing is modulated by antigen exposure. In explorative experiments, it was observed that splenic and peripheral blood CD8+ KLRG1
high, CD27
low, CD44
high, CD62L
low T cells respond to
in vitro stimulation with anti CD3 and
in vivo sporozoites infection by producing IFNg (see Additional file
1: Figure S1 and Figure S2). This is consistent with CD8+ KLRG1
high, CD27
low, CD44
high, CD62L
low T cells participating to protection by producing IFNg. However, these cells do not account for all the IFNg produced in response to
in vitro stimulation with anti CD3 (see Additional file
1: Figure S1 and Figure S2), or
in vivo stimulation with sporozoites see Additional file
1: Figure S1 and Figure S2). It is, therefore, conceivable that other CD8+ T cells with low expression of KLRG1 are generated during this infection/cure regimen. Such cells respond to sporozoite infection with IFNg production and display an elevated ability to respond to T cell receptor triggered by releasing IFNg. The role of this CD8+ T cell phenotype in protection remains to be determined. KLRG1 has been shown to down regulate T cell receptor signaling [
38,
39], thus suggesting that the acquisition of the KLRG1
high phenotype is important for effector memory population after antigen encounter and execution of effector function.