Alessandra Franco MD PhD UCSD School of Medicine Department of Pediatrics Division of Allergy Immunology and Rheumatology

Transcription

1 Immunodominant peptides derived from the heavy constant region of IgG1 stimulate natural regulatory T cells: identification of pan- HLA binders for clinical translation Alessandra Franco MD PhD UCSD School of Medicine Department of Pediatrics Division of Allergy Immunology and Rheumatology

2 Immune regulation is key in the immune homeostasis Immune regulation is key in the response to therapy in a large variety of immune-mediated diseases

3 Regulatory T cells phenotypically CD4+ CD25 high Two main human Treg lineages have been described 1) natural (n)treg are derived from the thymus during fetal life and recognize self peptides. 2) peripherally-induced (i)treg recognize not-self, arise from: a) naïve T cells under unique repeated stimulatory conditions (i.e. transforming grow factor (TGF-b) b) pro-inflammatory T cells (Th17, Th1, CD8+ cytotoxic T cells) following repeated antigenic stimulation (itreg are specific for the pathogen)

4 Kawasaki Disease: a disease model where immune regulation is key in the response to therapy

5 Kawasaki Disease (KD) is a self-limited T cell-mediated pediatric vasculitis of the coronary arteries. In San Diego County, the average incidence is 25/100,000 children <5 yrs. of age and approximately new cases are diagnosed and treated each year by the KD Team at Rady Children's Hospital San Diego If untreated, 25% of children will develop aneurysms of the coronary arteries. Treatment with high dose intravenous immunoglobulin (IVIG) reduces the rate of aneurysms to 5%

6 T helper 17 cells recruit CD8+ T cells (CTL) in the arterial walls α-sma α-sma αsma and IL17 Human Pathology 2012

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8 Understanding IVIG therapy and its success in Kawasaki disease: Fc-specific Treg?

9 Autoimmunity 2014 Fc-specific Treg expand only in sub-acute KD subjects with normal arteries after IVIG Subject 7 CD25 % CD4+CD25 high T cells Control 10µg/ml 100µg/ml 1µg/ml 10µg/ml CD subject 1 subject 2 subject 3 subject 4 subject 5 No Ag Fc µg/ml No Ag No Ag No Ag No Ag subject 6 subject 7 subject 8 subject 9 subject No Ag No Ag No Ag No Ag No Ag No Ag subject 11 subject 12 subject 13 subject 14 subject No Ag µg/ml Fc F(ab) 2 Dilated arteries or aneurysm No Ag No Ag No Ag µg/ml

10 Autoimmunity 2014

11 Fc-specific ntreg circulate in healthy donors but not adult KD patients

12 Hypothesis for the lack of Fc-specific ntreg Clonal deletion (mutated maternal Fc?) Clonal anergy HLA type/hla binding affinity of the relevant Fc peptides Antigen processing

13 The T cell receptor (TCR) recognizes a complex represented by MHC molecules and a peptide bound to the MHC binding groove

14 Antigen Processing and Presentation to MHC class II-restricted T Cells (CD4 + ): the data suggest that relevant Fc peptides are not presented to ntreg in KD patients that develop arterial complications

15 Fine specificity of ntreg: 64 peptides 15 amino acid long, 10 amino acid overlap

16 Method to define ntreg specificity PBMC separation Culture 4 days PBMC with 20µg/ml Fc peptide (97% pure) without exogenous IL-2 Read outs: 1. IL-10 measured in culture supernatants 2. CD4+ CD25 high T cell expansion by flow cytometry

17 Study population KD subjects after IVIG

18 IL10 responses to 6 immunodominant Fc peptides recognized by ntreg in KD subjects after IVIG Fc 51-65(Rank 1, 4/8, 50%) Fc (Rank 3, 2/8, 25%) Fc (Rank 2, 3/8, 37.5%) Fc (Rank 3, 2/8, 25%) Fc (Rank 2, 3/8, 37.5%) Fc (Rank 3, 2/8, 25%)

19 Fc position Sequence TAALGCLVKDYFPEP CLVKDYFPEPVTVSW YFPEPVTVSWNSGAL VTVSWNSGALTSGVH TFPAVLQSSGLYSLS LQSSGLYSLSSVVTV LYSLSSVVTVPSSSL SVVTVPSSSLGTQTY SVFLF PPKPKDTLMI PPKPKDTLMISRTPE TYRVVSVLTVLHQDW SVLTVLHQDWLNGKE NNYKTTPPVLDSDGS TPPVLDSDGSFFLYS QGNVFSCSVMHEALH SCSVMHEALHNHYTQ Autoimmunity 2015

20 Study population healthy adult donors

21 IL10 responses to 6 immunodominant Fc peptides recognized by ntreg in healthy adult donors Fc (Rank 1, 18/36, 50.0%) Fc (Rank 4, 14/36, 38.9%) Fc (Rank 2, 16/36, 44.4%) Fc (Rank 7, 11/36, 30.6%) Fc (Rank 3, 15/36, 41.7%) Fc (Rank 9, 9/36, 25%)

22 Binding affinity of 6 immunodominant Fc peptides to different DR, DQ and DP alleles of MHC class II DR alleles DQ alleles DP alleles Fc HLA binding Fc No binding Fc Fc No binding Fc Fc

23 Study population RA subjects

24 IL10 responses to 6 immunodominant Fc peptides recognized by ntreg in RA subjects Fc (Rank 1, 6/14, 42.9%) Fc (Rank 3, 3/14, 21.4%) Fc (Rank 2, 5/14, 35.7%) Fc (Rank 3, 3/14, 21.4%) Fc (Rank 2, 5/14, 35.7%) Fc (Rank 4, 2/14, 14.3%)

25 Promiscuous HLA class II binder Fc Promiscuous HLA class II binder Fc Promiscuous HLA class II binder Fc Fc (binds DRB1*12:01) Fc (binds DQB1*06:02) Fc (binds DRB1*07:01 and DRB1*04:05 )

26 ntreg expansion in response to peptide stimulation Healthy adult donors RA subjects In progress

27 Culture supernatant of a Fc specific ntreg line down-regulate IFNγ secretion by autologous pro-inflammatory T cells

28 ntreg responses to the Fc are different in healthy donors and RA patients P = , Mann-Whitney test

29 The phenotype of Fc-specific Treg clones indicate that these T cells are natural Treg (ntreg) Treg clones CTLA-4 GITR PDCD1 IL-10 IL-4 TGFβ Treg clones IL-17 FOXP3 CD45RA IL-7r IL-15r CCR6 CCR7 CCR4 CD25 CD4

30 IgG+ B cells and their role in expanding Fc-specific ntreg Treg expansion IL-10 B cell expansion? IL-4 HLA Class II TCR Treg A new model of B cell-t cell cooperation?

31 Fc-specific ntreg recognize autologous IgG+ B cells

32 live versus fixed IgG+ autologous B cells as antigen presenting cells Live autologous IgG+ B cells Fixed autologous IgG+ B cells Live autologous IgG+ B cells Fixed autologous IgG+ B cells IL-10 pg/ml IL-4 pg/ml Treg clones Treg clones IgG molecules on B cells need to be processed and presented by HLA molecules to stimulate Fc-specific Treg clones

33 Chemokine receptors suggest homing to the lymph nodes and proximity to the germinal center: representative Fc specific Treg line Anti-CD Events 42.3 Events Anti-CD4 Anti-CXCR5 Anti-CCR7

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35 Conclusions We identified the fine specificity of an important ntreg lineage involved in immune homeostasis in health and diseases: 3-5 immunodominant Fc peptides could serve in the clinic as the first approach to induce immune regulation CCR7 expression and B cell antigen presentation suggest that Fc-specific ntreg operates in the lymph nodes IgG+ B cells can activate Fc-specific ntreg that reside in the germinal centers (CXCR5+) Fc-specific ntreg secrete IL-4 in addition to IL-10 and promotes B cell survival/expansion This is the main mechanism of IVIG in some clinical settings: Fc peptides by-pass the need for antigen processing that is affected in the two disease models studied (KD and RA): Fc peptides offer an optimized approach to expand Fc-specific ntreg in vivo

36 Application of the technology Autoimmunity Vascular inflammation Prevention of miscarriages in autoimmune women Neurological disorders successfully treated with IVIG Kawasaki disease and other diseases where the IVIG mechanism resides in Immune regulation

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38 UCSD School of Medicine Department of Pediatrics AIR Division Li-En Hisieh Negar Benhamfar Ranim Touma Acknowledgments UCSD School of Medicine Department of Medicine RAI division Gary Firestein David Boyle Andre Matti La Jolla Institute for Allergy and Immunology Alessandro Sette John Sidney Kawasaki Disease Center Jane C Burns Adriana H Tremoulet Chisato Shimizu Joan Pancheri DeeAnna Sherrer