Platelets and their Beneficial and Detrimental Roles in Immunity JOHN W. SEMPLE PROFESSOR OF TRANSFUSION MEDICINE, LUND UNIVERSITY Talk Outline: 1.Platelets: general aspects 2.Platelets and immunity 3.Conclusions Platelets: general aspects Platelet Haemostatic Function: Resting Platelets Activated Platelets Advances in Platelet Biology 1998, Henn, platelet CD154 expression 1979, Ashi, others, platelet-bacterial interactions 2003, Elzy, platelets mediate adaptive immunity. 1971 Vane 1874 Osler Shows aspirin prevents Platelets are real 1918 Glanzmann platelet aggregation cells Describes “weak” 2008, Weyrich, others, platelet disease platelets differentiate and divide 1800 1900 2000 1842 Donne 1962 Born First describes platelet isolation 2004, platelets Semple, others, platelet TLR expression 1882 Bizzozero 1974 Nurden & Caen Names platelets GPIIb/IIIa associated with & popularizes platelet aggregation clotting function 1985 Stenberg 2005/6, 1906 Wright Platelet-derived P-selectin supports Kubes, Semple, others, Discovers that Megakaryocytes leukocyte adhesion platelet TLR is functional are the precursors of platelets Courtesy of AS Weyrich, adapted. Platelets are Immune cells. Kapur R et al. J Immunol, 2015, 194: 5579–5587 Why do platelets have immune properties? Evolutionary links of platelet immunity: Hemocytes Thrombocytes Platelets •Phagocytes (Mac.-like). •Aggregate and gel/clot plasma. •Aggregate and clot plasma. •Anti-bacterial peptides. •Phagocytic. •Phagocytic. •Express TLR/innate receptors. •Express TLR/innate receptors. •Express TLR/innate receptors. •Can aggregate and clot •Anti-bacterial peptides. •Anti-bacterial peptides. hemolymph at sites of injury. Invertebrates Vertebrates Mammals Levin J In: Platelets, 4th Ed., 2019 Evolutionary links of platelet immunity: Hemocytes Thrombocytes Platelets •Phagocytes (Mac.-like). •Aggregate and gel/clot plasma. •Aggregate and clot plasma. •Anti-bacterial peptides. •Phagocytic. •Phagocytic. •Express TLR/innate receptors. •Express TLR/innate receptors. •Express TLR/innate receptors. •Can aggregate and clot •Anti-bacterial peptides. •Anti-bacterial peptides. hemolymph at sites of injury. Invertebrates Vertebrates Mammals Levin J In: Platelets, 4th Ed., 2019 Evolutionary links of platelet immunity: Hemocytes Thrombocytes Platelets •Phagocytes (Mac.-like). •Aggregate and gel/clot plasma. •Aggregate and clot plasma. •Anti-bacterial peptides. •Phagocytic. •Phagocytic. •Express TLR/innate receptors. •Express TLR/innate receptors. •Express TLR/innate receptors. •Can aggregate and clot •Anti-bacterial peptides. •Anti-bacterial peptides. hemolymph at sites of injury. Invertebrates Vertebrates Mammals Levin J In: Platelets, 4th Ed., 2019 Evolutionary links of platelet immunity: Hemocytes Thrombocytes Platelets •Phagocytes (Mac.-like). •Aggregate and gel/clot plasma. •Aggregate and clot plasma. •Anti-bacterial peptides. •Phagocytic. •Phagocytic. •Express TLR/innate receptors. •Express TLR/innate receptors. •Express TLR/innate receptors. •Can aggregate and clot •Anti-bacterial peptides. •Anti-bacterial peptides. hemolymph at sites of injury. Invertebrates Vertebrates Mammals Levin J In: Platelets, 4th Ed., 2019 Is it possible?: Oyster Hemocytes Human Bronchial Macrophages (Rebelo MdF et al. PLoS ONE 8(2): e57384. doi:10.1371) (Creative Commons) Macrophage •Host Defense. •hemostasis (TF etc.) Hemocyte •Host Defense •Hemostasis Platelet Complexity •Hemostasis requires •Host defense (TLR etc.) specialization Platelets and Immunity Immune Regulation Direct Effects Anti-infectious P-selectin adhesion Effects Chemokine Recep. TLR 1-9 Allo. vs Autol. HLA Thrombocidins (Transplant./Transfusion) Β-defensins CD40/CD40L Neutrophil activation (T cell activation, Dendritic cell diff.) Cancer Autoimmunity Immune Resistance Effects Effects CD40/CD40L HLA transfer. Microparticles Microparticles IL1/IL8 Cytokines Platelet mass NK resistance Interaction between inflammation, hemostasis and the plaque: Wagner DD. Arterioscler Thromb Vasc Biol. 2005;25:1321-4. Adherence and Vascular smooth Inflammatory Adherence and Foam-cell aggregation of muscle cell cell activation entry of white blood cells formation platelets migration TRIM (Transfusion related immunomodulation): In 1999, Blajchman coined the term “Transfusion- Related Immunomodulation” (TRIM) in an attempt to unify the immunosuppressive effects of blood transfusions. Blajchman MA. Transfusion-associated immunomodulation and universal white cell reduction: are we putting the cart before the horse? Transfusion 1999;39:665-70. Resting Allogeneic Platelets turn off CD8+ T cells. Resting allogeneic platelets mediate TRIM: 108 BALB/c or SCID (H-2d) platelets weekly BALB/c or SCID skin graft CBA (H-2k) t Blood Collection Time to graft Pre- (anti-donor Abs) rejection. bleed Aslam et al. Transfusion. 48:1778-1786, 2008 Resting allogeneic platelets mediate TRIM: 108 BALB/c or SCID (H-2d) platelets weekly BALB/c or SCID skin graft CBA (H-2k) t Blood Collection Time to graft Pre- (anti-donor Abs) rejection. bleed Aslam et al. Transfusion. 48:1778-1786, 2008 Resting allogeneic platelets mediate TRIM: Non-transfused CBA Recipient (13 day post-transplant of Different BALB/c skin) allo-platelet transfusions CTL Non- transfused Skin MHC Rejection Platelet-transfused CBA Recipient (30 day post- transplant of BALB/c skin) Transfused allogeneic platelets Skin Rejection MHC Inhibited Aslam et al. Transfusion. 48:1778-1786, 2008 Upon Storage, Platelet bags accumulate Pro- and Anti- inflammatory agents: Cytokines (e.g. IL1, TGF, PF4) Serotonin HLA molecules Residual Leukocytes Oxidized Lipids Chemokines (e.g. CXCl1-7) CD40L Human soluble MHC class I from platelets and immunomodulation: Ghio et al Blood 93:1770, 1999 Puppo et al Int Immunol 12:195, 1999 Fournel et al J Immunol 164:6100, 2000 Spaggiari et al Blood 99:1706, 2002 Ghio et al Transfusion 48:1591, 2008 Using in vitro assays, soluble MHC class I molecules can induce immunosuppression via the induction of Fas-dependent apoptosis in CD8+ T cells and Natural Killer cells. Platelets are Immune cells. Kapur R et al. J Immunol, 2015, 194: 5579–5587 Activated platelets are more immunoregulatory than resting platelets: TREM-1 MHC Class I TLR 1-9 MHC Class I (denatured) (denatured) TLR 1,2,4-6 Resting Activated Platelet Platelet MHC Class I CD40L (Intact) CD40 T cell B cell and T cell Suppression Activation/Suppression Platelets are Immune cells. Kapur R et al. J Immunol, 2015, 194: 5579–5587 Activated Platelets release many immunomodulatory molecules: Cytokines IL-1β MHC class I (Inflammation) (CD8+ T cell interactions) TGF-β (immunosuppression) CD40L/CD40 Chemokines CXCL, CCL Activated (Cell recruitment) Platelet Lipid Mediators, 12-HETE (Inflammation) Thrombocidins (anti-microbial) β-Defensins PF4 (anti-microbial) Serotonin, 5-HT (Inflammation) November 2011 | Volume 7 | Issue 11 | e1002355 Kapur R et al. J Immunol, 2015, 194: 5579–5587 T cell activation is regulated by signals derived from the TCR/CD3/CD4 complex and the CD40L (CD154) and CD28/CTLA-4 co-stimulatory molecules: CD4+ T Cell Co-stimulatory signals Antigen specific TCR signals (- ) / [+] lck CD3 γ δ ε ζ ζ η η Cα Cβ α,β CD28/ Vα Vβ TCR CTLA-4 CD40L Peptide antigen CD40 CD4 MHC class II signal CD80 (B7.1)/ [+] CD86 (B7.2) Antigen Presenting Cell (APC) Platelet CD40L (CD154) expression and function in ITP: Blood. 2005;105:215-218 Patients with active disease Autoreactive B cell CD154 CD40 Activated Plt CD154 expression Ig GPIIbIIIa Anti-GPIIbIIa autoantibodies Kapur R et al. J Immunol, 2015, 194: 5579–5587 Activated platelets have the ability to stimulate CD8+ T cells: Mol Cell Proteomics 13:3308–3319, 2014. Platelets contain all the machinery necessary for cross presentation to CD8+ T cells: DenaturedDenatured MHC Class I + (plasma) Alpha granule α-granule PDI Platelets can cross present antigens to CD8+ T cells: Target Lysis (platelets and MK) CD8+ T cell CD40/80/86 CD154 Activated Plt MHC I expression TcR MHC Class I plasmodium peptide Activated murine MHC class I+ platelets present plasmodium peptides to CD8+ T cells and can act as a Ag delivery vehicles to protect against P. berghei induced fatal experimental cerebral malaria (ECM). PbA-OVA: Plasmodium berghei ANKA-OVA Can megakaryocytes (MK) cross-present antigen? Exogenous Protein Antigen Internalization APC CD8+ T cell Loading peptide onto MHC class I Presentation to CD8+ T cells MKs move OVA into endosomes, lysosomes and alpha granules. MKs load the OVA immunogenic peptide onto MHC Class I molecules Zufferey A et al. 1:1773-1785, 2017 While MKs process and present OVA, they are capable of transferring the MHC-OVA complexes to platelets. Malarial peptides Ovalbumin peptides Endogenous GPIIIa peptides Zufferey A et al. 1:1773-1785, 2017 Platelets MK MHC class I Proteasome Endosomes TAP Self Platelet Protein CD8+ Foreign Protein Lysosomes T cell Ag Zufferey A et al. Blood Adv. 1(20):1773-1785, 2017 Conclusions: Resting platelets tend to be immunosuppressive for adaptive immunity whereas activated platelets have the ability to turn on adaptive immune responses. Platelets express a variety of pro- and anti-inflammatory molecules that link them with autoimmune (ITP) and alloimmune (transfusions) regulation. Although platelets and MK are considered innocent bystanders and are the target of immune attack, evidence suggests that they can significantly influence Immune effector systems. Taken together, it appears that platelets and megakaryocytes have a critical role to play as infectious circulating sentinels and immunomodulators. Thank You Questions?.