Platelets present antigen in the context of MHC class I

Abstract

Platelets are most recognized for their vital role as the cellular mediator of thrombosis, but platelets also have important immune functions. Platelets initiate and sustain vascular inflammation in many disease conditions, including arthritis, atherosclerosis, transplant rejection, and severe malaria. We now demonstrate that platelets express T cell costimulatory molecules, process and present Ag in MHC class I, and directly activate naive T cells in a platelet MHC class I-dependent manner. Using an experimental cerebral malaria mouse model, we also demonstrate that platelets present pathogen-derived Ag to promote T cell responses in vivo, and that platelets can be used in a cell-based vaccine model to induce protective immune responses. Our study demonstrates a novel Ag presentation role for platelets.

Figure 1

Platelets Express Antigen Presentation Molecules. A) MHCI expression. Platelets from β2M^−/− and WT mice (MFI=Mean Fluorescent Intensity; n=4 ± S.D., *P<0.01 vs β2M^−/−). B) CD40. Resting or activated human platelets (2 µM TRAP or U46619; n=4 ± S.D., *P<0.01 vs IgG). C) CD86. Human platelets express CD86, which is increased with stimulation. D) CD86 Quantification (n=4 ±S.D.,*P<0.05, **P<0.01 vs IgG). E) Mouse platelets do not express CD86 (n=4 ± S.D.). F). ICOSL. Mouse platelets were incubated with control PBS or activated with thrombin before addition of control IgG or anti-ICOSL antibody (n=4 ± S.D., *P<0.05).

Figure 2

Platelets Provide T cell Co-Stimulation. T cells were incubated in anti-CD3 antibody or control PBS coated plates alone or with platelets or anti-CD28 antibody. A) IL-2 was measured by ELISA (n=4; ±S.D.,*P<0.01 vs CD3) and B) CD69 expression was measured by FACS (n=4; ±S.D.,*P<0.05 vs CD3).

Figure 3

Platelets Process and Present Antigen. A) Platelets present antigen. Platelets were incubated with control PBS, OVA peptide only (200 µg/mL), thrombin, or OVA peptide with thrombin stimulation (Thr=thrombin, 0.2 U/mL). MHCI-OVA complex was measured by FACS. Representative plots. B) Quantification (n=4 ± S.D.*P<0.01 vs OVA). C) Full Ovalbumin. Platelets were incubated with control buffer or ovalbumin with thrombin and OVA antigen presentation in MHCI determined at multiple time points (n=4 ± S.D. *P<0.05 vs Control).

Figure 4

Platelets Present Antigen to T cells. A) In Vitro. WT or OT-I T cells were incubated with control buffer or platelets from WT or OVA-Tg mice. IL-2 was measured by ELISA (n=4 ± S.D., *P<0.01 vs WT). B) INFγ (n=4 ± S.D., *P<0.01 vs WT). C) In Vivo. WT or OT-I mice were given WT or OVA-Tg platelets i.v. and 48 hrs later plasma was isolated to measure IL-2 by ELISA. (n=4, ±S.D., *P<0.01 vs WT).

Figure 5

Platelets Increase T cell Responses to P. berghei. Platelet depleted P. berghei infected mice have A) lower plasma IL-2 compared to control IgG treated infected mice (day 4 post infection, n=5 ± S.D., *P<0.01 vs IgG), and B) decreased IFNγ (n=4 ± S.D., *P<0.05 vs IgG). C) Platelets increase MHCI expression in ECM. Platelets were isolated from infected mice on day 6 post infection and MHCI expression determined by FACS. D) Quantification of MHCI^high expressing platelets (n=5 ± S.D., *P<0.01 vs Control Uninfected).

Figure 6

Platelets present Plasmodium derived antigen to T cells. A) Platelets acquire and present antigen. OT-I T cells were incubated alone, with iRBC, or with iRBC and WT or β2M^−/− platelets. 48hrs later T cell CD25 expression was determined by FACS. WT platelets increased OT-I T cell CD25 expression (n=4 ± S.D., *P<0.02 vs iRBC) and B) increased IL-2 production (n=4 ± S.D., *P<0.03 vs iRBC). C) Platelets were isolated from control mice or mice infected with PbA-OVA and MHCI-OVA measured by FACS (n=5 ± S.D., *P<0.05 vs Control).

Figure 7

Platelets Can Be Used as a Cell Based Vaccine. A) Platelets from WT and β2M^−/− mice were incubated with OVA peptide SIINFEKL (300 µg/mL), thrombin stimulated (0.2 U/mL), washed, and 1×10⁷ platelets injected i.v. into WT mice. This was repeated 10 days later, and platelet treated or control untreated mice were infected with PbA-OVA 21 days after the first platelet injection. Survival (n=5 ± S.D., *P<0.01 vs β2M^−/−). B) Representative day 7 post infection blood smear. C) Parasitemia quantification (n=5 ± S.D., *P<0.05, **P<0.01 vs WT). D) OVA specific T cells. Mononuclear cells were isolated from the spleens of mice on day 4 post-infection and the number of OVA specific T cells quantified by flow cytometry (n=5 ± S.D., *P<0.03; vs β2M^−/−).