Current Understanding of Immune Function & Transfusion

1. Mark W. Hall, MD Critical Care Medicine Nationwide Children’s Hospital, Columbus, OH Current Understanding of Immune Function & Transfusion

2. Disclosures • No financial disclosures • I will briefly mention off-label use of medications.

3. Objectives • Identify components of the innate and adaptive immune systems. • Describe the dynamic immune response in critical illness. • Understand the current evidence for the immunomodulatory nature of transfusion.

4. In other words…..does this patient need an anti-inflammatory therapy or a pro-inflammatory therapy? Does this patient have an over- or under-active innate immune response?

5. Pro-inflammatory IL-1β TNF IL-6 (?) IL-8 IL-17 Eicosanoids Anti-inflammatory IL-10 TGFβ IL-11 sTNF receptor IL-1ra IL-4 Cytokines LPS LTA PG MDP

6. The Cast of Characters – Innate immunity Neutrophil Monocyte • Innate immune cells: • Include PMNs, monocytes, macrophages, dendritic cells, NK cells • First line of cellular defense • Recognize broad classes of pathogens/molecules via cell-surface receptors (eg TLRs, FcR) • Response should be of the of same magnitude with each stimulation • Effectors: Cytokines and chemokines

7. The Cast of Characters – Adaptive immunity Lymphocyte • Adaptive immune cells: • Include T and B lymphocytes • Typically require antigen presentation • Perpetuate and modulate the immune response • Response is highly antigen-specific, memory • Effectors: Cytokines and chemokines, Ab (opsonization)

8. It’s not just about quantity….. • CD4+ T cells • TH1 • Proinflammatory • IL-2, IFNγ, GM-CSF • Induce B cells to make IgG • TH2 • Anti-inflammatory • Autoimmune • IL-10, TGFβ, IL-4

9. Regulatory T cells (Treg) • Ultra immunosuppressive CD4+ T cells. • Appear to be resistant to the wave of apoptosis that affects other T cells in sepsis. • By Day 5, are the most prevalent type of T cell in adult sepsis. patients. • Perpetuate an anti-inflammatory phenotype?

10. Pro-inflammatory IL-1β TNF IL-6 (?) IL-8 IL-17 Eicosanoids Anti-inflammatory IL-10 TGFβ IL-11 sTNF receptor IL-1ra IL-4 Cytokines LPS LTA PG MDP

11. Pro-inflammatory IL-1β TNF IL-6 (?) IL-8 IL-17 Eicosanoids Yeah….but therapies targeting these mediators have failed to consistently improve sepsis outcomes! Anti-LPS IL-1ra Anti-TNFα* Anti-bradykinin Anti-PAF Methylprednisolone/ dexamethasone Cytokines LPS LTA PG MDP

12. Monneret, Adv in Sepsis, 2005

13. Serial vs. simultaneous Function Goal: Restore immune responsiveness while reducing systemic inflammation. Plasma

14. Where can we look for markers of immunoparalysis?

15. 2. TNF 3. 1. Healthy Monocyte 1. Phagocytosis 2. Intracellular killing 3. Antigen presentation 4. Extracellular TNF production TNF 4. TNF TNF

16. Immunoparalysis HLA-DR expression < 30% TNF Immunoparalyzed Monocyte TNF TNF TNF

17. Immunoparalysis TNF Immunoparalyzed Monocyte TNF TNF Impaired ex-vivo LPS-induced TNF production TNF

18. Immunoparalysis in the PICU Immunoparalysis (ex vivo LPS-induced TNFα production capacity < 200 pg/ml) in critically ill children with MODS: Relative risks (RR) with 95%CI if immunoparalysis is present for more than 3 days: OutcomeRR with 95%CI New Infection 3.3 (1.8 – 6.0) Death 5.8 (2.1 – 16) Hall et al, Intensive Care Medicine 2011

19. Pediatric MODS patients who developed nosocomial sepsis (■) had lower ex vivo TNFα production over time compared to those without nosocomial sepsis (▲).

20. Pediatric MODS patients who died (■) had lower ex vivo TNFα production over time compared to those who survived (▲).

21. Ex vivo TNF and Monocyte HLA-DR • These two measures of innate immune function tend to track together…. Hall et al, Intensive Care Medicine 2011

22. Early TNFα Production Capacity and 14-day Nosocomial Infection n = 200 (43 infections) Nofziger, CCM 2011

23. Early TNFα Production Capacity and 28-day Mortality n = 200 PRISM III: AUC: 0.87 PELOD: AUC: 0.80 (9 deaths) Nofziger, CCM 2011

24. PICUFlu Sites in the LPS-stimulation Study

25. “Cytokine storm” Hall et al, CCM 2013 • High levels of both pro- and anti-inflammatory mediators can be found in the serum……the response is often mixed, but these are not functional measures.

26. The innate immune response and death Hall et al, CCM 2013 • Critically ill children who died (n = 8) had lower ex vivo LPS-induced TNFα production capacity than did children who survived (n = 44).

27. OK…so the lymphocyte is important too! Hotchkiss, J Immunol, 2001

29. Immunoparalysis without initial infection? • Immunoparalysis has been reported in the setting of: • Pancreatitis • Ho, Am J Gastro, 2006 • Cardiopulmonary bypass • Allen, CCM, 2006 • Cornell, JTCVS, 2011 • Trauma……

30. Trauma at Nationwide Children’s Hospital Early reductions in immune function predict the development of infection in critically injured children.

31. So what?

32. Reversibility of Immunoparalysis • Case series and a few small RCT suggest that critical illness-induced innate immune suppression is reversible • Interferon-γ • GM-CSF • The cancer literature is beginning to suggest that we may be able to target adaptive immune dysfunction as well…. • Anti-PD-1 therapy • rhIL-7 • Real-time immune monitoring is becoming feasible. • Immunophenotype-tailored management plans

33. n = 19 subjects per group.

35. There were no nosocomial infections in the GM-CSF treated group. All patients in the standard therapy group developed secondary infection.

36. What contributes to immune dysfunction in critical illness?

37. Unintended Immunomodulation

38. Transfusion-related immunomodulation Transfusion is still one of the most commonly employed therapies in the PICU.

39. Transfusion-related immunomodulation • Some sequelae of transfusion appear to impair the immune response: • In the 1970s, lower rejection rates were observed in previously-transfused renal transplant patients. • Microchimerism / tolerance • Increased rate of bacterial infection • Some sequelae of transfusion, however, have a pro-inflammatory phenotype: • Febrile non-hemolytic transfusion reactions • Transfusion-related acute lung injury

40. What’s in the bag? Lannan et al, Blood Cell Mol Dis, 2012 • Pro-inflammatory mediators: • Anti-inflammatory mediators: • Apoptotic leukocytes, IL-10, TGFβ promote regulatory T cells?

41. Leukoreduction • Donor leukocytes were strongly implicated in TRIM. • Pre-storage leukoreduction • Removes the vast majority of donor WBC • Reduced FNHTR, CMV transmission, HLA sensitization • As much as 83% reduction in TRALI (Blumberg et al, Transfusion, 2010) Only transfused subjects Blumberg et al, Transfusion, 2007

42. What’s still in the bag – Lipids • Silliman et al, Transfusion, 2011 • Nonpolar lipids accumulate in stored, leukoreduced RBC • Two-hit rat model of ALI (i.p. LPS + i.v. lipid infusion) • Increased superoxide production • Increased pulmonary capillary leak

43. What’s still in the bag – Microparticles • Belizaire et al, J Am CollSurg, 2012 • Microparticles accumulate in stored, leukoreduced RBC • When concentrated and infused in a hemorrhagic shock model, MP induced lung inflammation

44. Neal et al, Transfusion Medicine Reviews, 2013 What’s still in the bag – Iron • Iron promotes inflammation in innate immune cells. • Bacteria love to eat iron. Not sure what happens when you give iron, lipids, MP etc. to an immunoparalyzed cell…

45. Murine allogeneic, leukoreduced transfusion model: • IV LPS was administered immediately prior to transfusion of fresh vs 14day stored RBC. • Plasma cytokines measured at 24hrs. • Older blood exacerbated the systemic inflammatory response.

46. Iron chelation (DFO, FO) at the time transfusion reduced the inflammatory response to transfusion of stored blood. What about washing?

47. Innate pro-inflammatory cytokines in the units. Innate pro-inflammatory cytokines in the mice.

48. Ok, so pre-storage leukoreduced blood can still induce systemic inflammation, but what about immune function?

49. In in vitro monocyte co-culture experiments, PRBC stored for 14 and 21 days were more immunosuppressive than 7-day old PRBC.

50. Suppression of LPS-induced TNFα production, but not IL-10 production also occurred at the mRNA level. • CPD-only RBC were immunosuppressive regardless of storage age. • Adsol-containing units were most immunosuppressive at Day 21. • Preservative solutions by themselves were did not impair TNF production capacity. • Transwell experiments (preventing cell-to-cell contact) also demonstrated suppression. • Small, soluble factor(s): Lipids? MP? Iron? miRNA?