1. A Role for Immature Myeloid Cells in Immune Senescence Elena Y. Enioutina Diana Bareyanand Raymond A. Daynes

2. Introduction  Immune senescence Causes and consequences of immune system aging in mammals Compromised innate & adaptive immune responses Oxidative stress & inflammatory cytokines  Myeloid cells Possessing immune suppressive activities NO-dependent mechanism Abnormal accumulation->cancers, persistent bacterial or viral infections, or after surgical trauma or thermal injury Gr1 + CD11b + cells as myeloid-derived suppressor cells (MDSCs)  MDSCs suppress the proliferation of both CD4 + and CD8 + T cells increased activities of arginine metabolizing enzymes, inducible NO synthase (iNOS), and/or arginase 1 (ARG-1)

3. Review Molecular mechanisms regulating myeloid-derived suppressor cell differentiation and function Trends in Immunology Volume 32, Issue 1 January 2011, Pages 19-25

4. I.immune-compromising activities of Gr1 + CD11b + cells contribute to immune senescence? II.characterize the signaling defects responsible for their immune suppressive activities in aged hosts? Introduction

5. The blood, secondary lymphoid organs, and BM of aged mice possess elevated numbers of myeloid cells bearing the Gr1 + CD11b + phenotype

6.  Gr1 + CD11b + cells residing in multiple lymphoid organs increased in aged mice ring shape : immature 5-fold similar 3 mo 18~22 mo CD11b+ BALB/c or DO11.10 3 mo 18~22 mo CD11b + Ly6C int Ly6G int -> CD31+(PECAM-1)

7. Mice carrying the MHC class II restricted rearranged T cell receptor transgene, Tg(DO11.10)10Dlo, react to ovalbumin (OVA) peptide antigen Intraperitoneal administration of OVA to transgenic mice results in a rapid deletion of the immature CD4 + CD8 + TCR lo thymocytes with progression to mature thymocytes BALB/c-Tg(DO11.10)10Loh/J

8. increased accumulation of immature myeloid cells in multiple secondary lymphoid organs of aged mice -> orchestrate some of the immune dysfunctions associated with advanced age

9. Stimulation of aged splenocytes with TLR4 ligand results in a significant increase in the percentage of Gr1 + CD11b + and CD4 + T cells producing proinflammatory cytokines

10.  Gr1 + CD11b + cells and CD4 + T cells in SPLs of aged mice produce proinflammatory cytokines 3 mo 22 mo Splenocytes : 1 × 10 6 cells/ml in the presence of 0, 10, or 100 ng/ml LPS A. Gr1 + CD11b + cells of BALB/c 6 h 24 h B. CD4 + cells of DO11.10 24 h C. Splenocytes culture of BALB/c 48 h Sup.-> ELISA Neutralizing

11. Gr1 + CD11b + cells from aged mice suppress CD4 + T cell responses, markedly increase iNOS or ARG-1 expression after activation, and produce elevated levels of proinflammatory cytokines

12.  Gr1 + CD11b + cells isolated from the SPLs of aged mice produce proinflammatory cytokines, increased activity of iNOS and ARG-1, suppressed CD4 + T cell proliferation 4 d Splenocytes DO11.10 mice (3 mo) 2 × 10 6 cells/ml+100 μg/ml OVA Gr1 + CD11b + cells from SPLs or BM of BALB/c : OVA-specific CD4 + T cell proliferation B. Gr1 + CD11b + cells isolated from the SPLs C. Gr1 + CD11b + cells isolated from the SPLs 24 h 48 h 24 h 48 h Sup. 48 h ELISA Sup.

13. Gr1 + myeloid cells residing in the lymphoid organs of aged mice negatively affect Ag-induced CD4 + T cell proliferation in vitro and the ability to mount T cell-dependent Ab responses in vivo

14.  Depletion of Gr1 + cells from the secondary lymphoid organs of aged mice normalizes their immune responsiveness A. Splenocytes DO11.10 mice (3 mo, 20 mo) 2 × 10 6 cells/ml+100 μg/ml OVA or Gr1 depleted splenocytes ( 20 mo ) Sup. In vivo Gr1 depletion of Gr1 by i.p. with anti-Gr1 mAb to aged DO11.10 mice on days −6, −3, and 0 Day 0 s.c with 50 μl of vaccine containing 50 μg OVA in Alum OVA-specific IgG Abs by ELISA T cell-dependent Ab responses

15. Activated Gr1 + CD11b + cells from aged donors express decreased levels of phosphorylated Akt and fail to inactivate GSK3β effectively

16. Int J Biol Sci 2010; 6:9-50 Review An Overview of Stress Response and Hypometabolic Strategies in Caenorhabditis elegans: Conserved and Contrasting Signals with the Mammalian System

17.  Purified Gr1 + CD11b + cells from aged donors have a reduced ability to phosphorylate Akt and are incapable of effectively inactivating GSK3β Gr1 + CD11b + cells isolated from SPLs of BALB/c mice cocultured with LPS (10 ng/ml) for 30 and 60 min Total cellular proteins prep. ->Western blot Total cellular proteins prep. ->Western blot decreased SHIP1 is only expressed by hematopoietic cells and inhibits the downstream activation of Akt by enzymatically converting PI3K- induced PIP3 to PIP2

18.  The inhibition of GSK3β activity in Gr1 + CD11b + cells from aged mice ameliorates the proinflammatory condition and reduces inducible iNOS activity. GSK3 inhibitor Gr1 + CD11b + cells isolated from SPLs of BALB/c mice cocultured with LPS (10 ng/ml) + IFN-γ or IL-4 reduced 48 h -> ELISA No change

19.  Treatment of activated CD4 + T cells isolated from aged donors with a specific GSK3 inhibitor enhances T cell proliferation, increases IL-2, and reduces IFN-γ production Splenocytes from DO11.10 mice were cultured 5 × 10 6 cells/ml with OVA (100 μg/ml). 4 d -> ELISA CD4 + T cells isolated from SPLs of DO11.10 mice (5 × 10 6 cells/ml) -> anti-CD3ε (2 μg/ml) coated tissue culture plates -> Adding anti-CD28 Abs (2 μg/ml) 24 h -> ELISA

20. Discussion Multiple lymphoid organs of healthy aged mice harbor increased numbers of Gr1 + CD11b + cells CD11b + Ly6C int Ly6G int -> suppressive activities of splenic Gr1 + CD11b + cells in aged mice ( Vitamin E -> enhancement ) MDSC -> ↑iNOS (Salmonella infected), ↑ ARG-1(transplantable tumor bearing) Monocytes undergo a microenvironment-dependent polarization process LPS + IFN-γ -> M1 macrophages -> iNOS IL-4, IL-13, IL-10 -> M2 macrophages -> ARG-1 immature Gr1 + CD11b + cells LPS + IFN-γ -> iNOS LPS + IL-4 -> ARG-1 Many other substances (e.g., GM-CSF, G-CSF, PGs, S100A8/A9 proteins, IL-1β, IL-6, and IL-12) have been implicated in the expansion and activation of MDSCs S100A8 and A9 are increased 1.5- to 2-fold in healthy aged mice NO produced by splenic Gr1 + cells is responsible for suppressing CD4 + T cell activities with aging Stimulated Gr1 + CD11b + cells from the SPLs of aged animals had a diminished capacity to activate Akt, resulting in a reduced ability to inactivate the downstream enzyme, GSK3β SHIP-1 and PTEN are enyzmes that inhibit the Akt activation Inhibition of GSK3 activity in stimulated Gr1 + CD11b + cells from aged mice decreased the production of proinflammatory cytokines and iNOS activity no effect on inducible ARG-1 activity Increase T cell proliferation

21. 3 rd IDO GVHD day 94 4 th IDO GVHD day 70 5 th IDO GVHD day 49 B6 vs IDO KO -> BALB.B B6 vs IDO KO -> B6 Discussion

22. 3 rd IDO GVHD day 94 4 th IDO GVHD day 70 5 th IDO GVHD day 49 B6 vs IDO KO -> BALB.B B6 vs IDO KO -> B6 Discussion

23. 3 rd IDO GVHD day 94 4 th IDO GVHD day 70 5 th IDO GVHD day 49 B6 vs IDO KO -> BALB.B B6 vs IDO KO -> B6 Discussion