Lectins: selected topics 3/2/17

Transcription

1 Lectins: selected topics 3/2/17

2 Selected topics Regulation of T-cell receptor signaling Thymic selection of self vs. non-self T-cells

3 Essentials of Glycobiology Second Edition

4 Signaling pathways associated with the BCR CD22 is actually a transmembrane receptor that inhibits BCR signaling. CD22 interactions with BCR prohibit the BCR from patching and aggregating into membrane signaling domains. CD22 also contains ITIM motifs in its cytoplasmic tail, which attenuate the activation of the BCR ITAM motifs by recruiting phosphatases (SHP-1) that dephosphorylate Igα/β.

5 CD22 is a member of the SIGLEC family of carbohydrate binding proteins (lectins) that bind sialic acid (SIGLEC 2) Relative balance between CD22-CD22 interactions and CD22-BCR interactions can determine the volume of the signal coming from a ligated BCR. CD22-BCR interactions may also interfere with effective antigen binding to the BCR. Altered protein sialylation would be predicted to modulate CD22 s ability to regulate BCR signaling.

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9 Glycan regulation of T-cell receptor signaling, conceptual similarities with regulation of B-cell receptor signaling

10 Functional TCR signaling complex Variable region, interaction with MHC CD3 co-receptor complex, comprised of ε, γ, δ subunits, contains cytoplasmic ITAM motifs TCR has small, inactive cytoplasmic domain ζ Subunits also possess ITAM motifs for recruiting kinases

11 T cells express either CD4 (helper T) or CD8 (cytotoxic T) as enhancing co-receptors CD4 is said to restrict a CD4+ T-cell so that it can only interact with the Class II type of MHC. Likewise, CD8+ T-cells are restricted to MHC Class I. The co-receptors enhance signaling by stabilizing the TCR-MHC complex, allowing prolonged signal propagation if there is a match between the TCR and the MHC class. CD4 and CD8 also recruit kinases to the signaling complex (Lck).

12 CD45 is a receptor protein tyrosine phosphatase (rptp) that enhances TCR signaling by removing an inhibitory phosphate from Lck Tyr-K. CD45 is a large protein with two PTP domains and extensive glycosylation. CD45 modulates T-cell signaling

13 CD45 splice forms interact differently with TCR complex Long form has less PTPase activity and does not stimulate TCR complex signaling effectively. Expressed in Naïve, quiescent T-cells. Short form has full PTPase activity and is expressed in activated T-cells (or previously activated cells that have become memory cells).

14 CD45 possesses a large extracellular domain that is highly glycosylated The three domains spliced out to make the short form (A,B,C) contain many sites of addition for O-linked glycans. Earl, L.A., (2008) Immunology and Cell Biology 86, 608

15 Many, but not all, O-linked glycans can be bound by a family of carbohydrate binding proteins (lectins) called Galectins. Galectin cross-linking clusters CD45 and shuts off its PTPase activity, generating a silenced TCR complex. Thematically, this is similar to CD22 and the BCR, except CD22 binding to α2,6 sialic acid enhances BCR signaling. Modified from Earl, L.A., (2008) Immunology and Cell Biology 86, 608

16 The appropriate glycosylation must be placed on CD45 in order to allow Galectin cross-linking On in active cells ST3Gal-1 is one of many On in naïve cells sialyltransferases that are upregulated by pro-inflammatory cytokines C2GnT=core 2 GlcNAc T ase, which is upregulated by antiinflammatory cytokines Earl, L.A., (2008) Immunology and Cell Biology 86, 608

17 Thymic selection, glycan mediated regulation of thymocyte maturation

18 Self vs. non-self selection

19 Thymic selection

20 Thymic involution is a normal component of aging

21 The thymus has a lobular architecture with trabeculae providing entry points to each lobe

22 Distinct regions can be discerned within each lobe

23 Extensive network of thymic epithelial cells provides large surface area for interaction with thymocytes

24 T-cell maturation and selection begins in the thymic cortex Immature T-cells arrive from the bone marrow and enter the thymus at the cortico-medullary junction as double negative cells, lacking expression of both CD4 and CD8, as well as a functional TCR. TCR rearrangement occurs in cortical thymocytes, producing receptors that may or may not recognize MHC displayed on cortical thymic epithelial cells. Thymocytes become double positive cells, expressing both CD4 and CD8.

25 T-cell maturation and selection begins in the thymic cortex Thymocytes that generate TCRs capable of recognizing MHC-I shut down expression of CD4 and thymocytes that generate TCRs capable of recognizing MHC-II shut down expression of CD8. Singly positive CD4+ and CD8+ thymocytes are attracted to the cortico-medullary junction by chemokines produced by macrophages and dendritic cells located in the medulla.

26 Double positive cells in the Cortex, Single positive cells in the Medulla Green: anti-cd4 Blue: anti-cd8 Red: Medullary thymic epithelial cell marker Takahama, Y. (2006) Nature Reviews Immunology 6, 127

27 Thymic selection for self vs. nonself requires two steps Step 1: Positive selection for thymocytes expressing rearranged TCRs that bind self-mhc presenting self-peptide on cortical thymic epithelial cells. Cells that can interact with MHC survive, those that can t apoptose; a positive survival signal. Cells that interact very strongly with self MHC and self-peptide are retained in the cortex and are eventually killed. Step 2: Negative selection for thymocytes that express rearranged TCRs that bind self-peptides presented by self-mhc on medullary epithelial cells, macrophages, or marrow-derived dendritic cells. Cells that interact strongly are killed, cells that do not interact or interact weakly are matured into CD4+ or CD8+ T-cells.

28 Thymic cortical epithelial cells express galectin 1 anti-galectin 1 anti-cytokeratin Epithelial marker anti-galectin 1 Baum, LG, et al. (1995) J Exp. Med. 181, 877

29 Core-2 synthesis generates ligands for Galectins Core2 GlcNAcT is upregulated in cortical thymocytes

30 Galectin 1 induced apoptosis of T-cell lines requires Core-2 O-glycans Core2 GlcNAcT mrna Time in culture + Galectin 1 1B11 recognizes Core 2 O- glycan on CD43 Galvan, M, et al. (2000) JBC 275, 16730

31 C2GlcNAcT activity in relation to thymic selection C2GlcNAcT expression by thymocytes in the thymic cortex increases with residence time and decreases as the differentiating T-cell migrates to the medulla Strong TCR activation by self-bound MHC results in retention of the thymocyte in the cortex and maintenance of C2GlcNAcT activity (negative selection) Weak TCR activation results in migration of the T-cell to the thymic medulla and down-regulation of C2GlcNAcT activity (positive selection) C2GlcNAcT activity generates substrates for Galectin binding, enhancing signaling through receptors that lead to apoptosis of cortical T-cells with self-specificity

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33 Paper for discussion

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