Enhancement of dendritic cell activation via CD40ligand-expressing γδ T cells is responsible forprotective immunity to Plasmodium parasites

1. Shin-Ichi Inoue, Mamoru Niikura, Satoru Takeo, Shoichiro Mineo, Yasushi Kawakami, Akihiko Uchida, Shigeru Kamiya, and
Fumie Kobayashi received for review March 15, 2012 ; approved July 24, 2012 , PNAS vol.109 12129-12134
Presented by
Shaheda Parveen
Msc. Biotechnology
3rd semester
Jamia Hamdard

2.  To elucidate the mechanism of γδ T-cell–associated protective
immunity against blood-stage malaria.
 Using γδ T-cell depletion by anti-TCRγδ mAb, found that γδ T cells
are necessary for the elimination of P. berghei XAT parasites during
the early stages of infection.
 using γδ T-cell– deficient (TCRδ-KO) mice, showed that γδ T cells
enhance DC activation through CD40/CD40L signaling during the
early stages of infection.
 The function of γδ T cells is to induce Th1cell differentiation and
increase the number of phagocytes, resulting in clearance of P.
berghei XAT.

3.  Malaria is one of the most serious
public health problems worldwide, .
 Plasmodium,the malaria-causing
protozoan parasite, has a complex life
cycle
 1- liver stage
 2- blood stage
Lifecycle of Plasmodium parasites
Inoue et al. (2013) Roles of IFN- γ and γδ T cells in protective
immunity against blood stage malaria ;frontiers in immunology
, review

4. αβ T cell
•αβ T cells, such as CD4+ helper T cells and CD8+ killer T cells, are typically related to
adaptive immunity.
•CD4+ αβ T cells regulate expansion of γδ T cells.
γδ T cell
•function as innate immune cells, the first line of defence against infectious pathogens
•In humans and mice, the number of γδ T cells in peripheral blood and spleen increases
after infection with malaria parasites. Bakir HY, et al. (2006)
•experiments have demonstrated that γδ T cells recognize malaria antigens. Therefore, γδ T
cells may have protective or pathological effects on blood-stage malaria. Dieli F, et al. (2001)
•γδ T cells respond immediately to Plasmodium infection, produce proinflammatory
cytokines, and facilitate the activation of other immune cells by responding directly to
malaria parasites.
TCR is a molecule found on the surface of T lymphocytes that is
responsible for recognizing antigens bound to major histocompatibility
complex (MHC) molecules.

5.  produced mainly by lymphocytes,such as αβ T cells,natural killer(NK)cells
and γƍ T cells induce by IL-12 and IL-18 from APCs such as DCs.
 An important pro inflammatory cytokine and a mediator of immune
responses against intracellular bacteria and viruses.
 enhances phagocyte activity, resulting in the elimination of extracellular
bacteria and protozoan parasites.
 Th1 cells that produce IFN-γ are critical for protective immunity. IFN-γ
activates phagocytes and IgG2a production from B cells, thereby clearing
P. berghei XAT parasites.

6. Robert H. Vonderheide (2007) Prospect of
Targeting the CD40 Pathway for Cancer Therapy
Clin Cancer Res ;13:1083-1088.
Member of the TNFR family, is expressed by B
cells, professional antigen-presenting cells, as
well as non-immune cells and tumors.
CD40 binds its ligand CD40L, transiently
expressed on T cells and other non-immune
cells under inflammatory conditions.
CD40L/CD40 interactions exert profound effects
on DCs, B cells, and endothelial cells
CD40 engagement on the surface of DCs
promotes their cytokine production

7.  considered to be important IFN-γ producers in blood-stage malaria
infections and to be associated with the control of malarial parasites.
 In the early stages of malaria infection, γδ T cells directly recognize
the pathogen through MHC-independent mechanisms that involve
the γδ TCR, and high levels of IFN- γ production and proliferation are
induced
 Proliferation depends on IL-2
 In P.falciparum , shown that inspite of γδ T cell , Nk cells were minor
IFN- γ producers in response to iRBCs before and after infection.

8. Inoue et al. (2013) Roles of IFN- γ and γδ T cells in protective
immunity against blood stage malaria ;frontiers in immunology
, review

9. Inoue et al. (2013) Roles of IFN- γ and γδ T cells in protective
immunity against blood stage malaria ;frontiers in immunology
, review

10.  Result-1
Protocol: inoculated P. berghei XAT infected iRBCs into
TCRδ-KO mice and control WT mice C57BL/6.
A. Role of γδT Cells on P. berghei XAT
B. due to higher in parasitemia TCRδ-KO
mice will die wrt. Control
C. Depleted CD4+ by sequential
administration of anti-CD4 mab after day 9
p.i. show higher parasitemia and died
after 30 day p.i.
D. Survival graph of fig. C
A , C - time coyrse analysis of parasitemia
B , D – survival curve

11.  Result-2
After Plasmodium infection, naive CD4+ T cells
(Th0 cells) differentiate into a large number of
Th1 cells that produce IFN-γ.
A. Zebra plots represent CD3+- and CD4+-gated
spleen cells from WT mice and TCRδ-KO mice.
B. Proportions of IFN-γ–expressing CD4+ T cells in
spleens from WT mice and TCRδ-KO mice.
C. Absolute number of IFN-γ– expressing CD4+ T
cells in spleens fromWT mice and TCRδ-KO mice
D. Estimation of the number of CD4+ T-cells
expressing IFN-γ in spleens from each mouse
group
E. IFN-γ concentration in the supernatant of
cultivated splenocytes from each mouse group
F. IFN-γ concentration in plasma from each mouse
group

12.  Result-3
A. B. The number of conventional DCs (cDC; CD11c+
B220− CD19− CD3−) and plasmacytoid DCs (pDC; in
spleens of WT mice increased transiently on day 5 p.i.
C. expression of indicators in cDCs and pDCs in spleens from
WT mice after infection
D. IL-12 levels in the supernatant of spleen cells cultivated with
P. berghei XAT antigens.
E. Intracellular cytokine assay of cDCs and pDCs in spleens .
F. Proportion of cDCs and pDCs in spleens from each mouse
group expressing IL-12
G. Estimation of the number of cDCs and pDCs expressing IL-12
in spleens from each mouse group p.i.

13.  Result-4
A. Absolute numbers of γδ T cells in spleens from
WT mice.
B. (Left) CD3+-gated splenocytes in WT mice
showing the proportion of γδ T cells in CD3+
lymphocytes. (Right) CD3+- and TCRγδ+-gated
spleen cells in WT mice showing the proportion
of γδ T cells expressing IFN-γ.
C. Proportions of γδ T cells expressing IFN-γ in
spleens from WT mice
D. Splenic sections in naive WT mice

14.  Result-4
E. Splenic sections in WT mice on day 5 p.i.
F. Proportions of γδ T cells adhering to DCs in splenic
sections of naive WT mice and WT mice on day 5 p.i.
G. Expression profiles of the CD40L molecule in CD4+ T
cells and γδ T cells in spleens (Sp) and peripheral
blood (PB) from WT mice.
Suggest that many γδ T cells adhere to DCs in the
spleens of both uninfected and infected mice and
that the proportion of adhering cells is increased by
the infection.

15.  Result-5
 To examined whether in vivo stimulation of CD40 by agonistic antibodies
prevented TCRδ- KO mice from failing to clear the parasites.
 TCRδ-KO mice were infected with P. berghei XAT and then were injected i.v.
with anti-CD40 agonistic mAb for stimulation of CD40 on day 4 p.i., 1 d before
DC activation.
 TCRδ-KO mice were injected with nonspecific control rat IgG on day 4 p.i as
control
 70% of TCRδ-KO mice treated with anti-CD40 agonistic mAb controlled
parasitemia levels and survived
 all control TCRδ-KO mice treated with rat IgG had high parasitemia and
eventually died
 Result

16. α It was hypothesized that γδ T cells would begin to play their roles in protective immunity from
day 10 p.i.
α However, an unexpected finding was that depletion of γδ T cells from day 9 p.i. had no effect
on the clearance of P. berghei XAT. In contrast, even from day 9 p.i., CD4+ T cells were needed
to clear the parasites.
α These results suggest that CD4+ T cells indeed were the effector cells of protective immunity
against P. berghei XAT and that marke expansion of γδ T cells is not required for protective
immunity against these parasites.
α Artificial stimulation of CD40 signaling by administration of agonistic anti-CD40 mAb to
TCRδ-KO mice enhanced protective immunity against P. berghei XAT parasites. These result
strongly suggest that γδ T cells play a crucial role in the stimulation of CD40 signaling in DC
through CD40L expression.
 Infection of TCRδ-KO mice with Plasmodium chabaudi leads to exacerbation of parasitemia,
although infected TCRδ-KO mice can clear the parasites eventually. Thus, dependence on γδ
T-cell–mediated protective immunity differs among Plasmodium species.
 However, in human cases, the number of γδ T cells is increased in the blood and spleen of
patients with P. falciparum . Therefore, γδ T cells play a critical role In protective immunity in
humans.
 FUTURE STUDIES : should examine whether human γδ T cells express CD40 after stimulation
with P. falciparum- infected RBCs. Previous results provide evidence for γδ T-cell– and DC-
mediated regulation of malaria via CD40 signaling and offer insights into immunotherapeutic
approaches for malaria, such as the use of specific chemicals for activating DCs via CD40
signaling.

17.  Bonneville M, O’Brien RL, Born WK (2010) γδ T cell effector functions: A blend of innate programming
and acquired plasticity. Nat Rev Immunol 10:467–478.
 Dieli F, Troye-Blomberg M, Farouk SE, Sireci G, Salerno A (2001) Biology of γδ T cells in tuberculosis and
malaria. Curr Mol Med 1:437–44
 Inoue et al. (2013) Roles of IFN- γ and γδ T cells in protective immunity against blood stage malaria
;frontiers in immunology , review
 Shin-Ichi Inouea, Mamoru Niikuraa, Satoru Takeoa, Shoichiro Mineoa, Yasushi Kawakamib, Akihiko
Uchidab, Shigeru Kamiyaa, and Fumie Kobayashia, (2012) Enhancement of dendritic cell activation via
CD40 ligand-expressing γδ T cells is responsible for protective immunity to Plasmodium parasites PNAS